Analgesic Compounds, Compositions, and Uses Thereof

ABSTRACT

The invention relates to compounds, compositions, and methods for diminishing pain in a subject in need thereof comprising administering the compounds and compositions herein described.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is entitled to priority under 35 U.S.C. §119(e)to U.S. Provisional Applications No. 61/312,482, filed Mar. 10, 2010 andNo. 61/378,781, filed Aug. 31, 2010, which applications are herebyincorporated by reference herein in their entireties.

BACKGROUND OF THE INVENTION

Pain is defined as an unpleasant sensory and emotional experience.However, pain can be informative and useful. For example, nociceptivepain is often indicative of injury (e.g., tissue damage) and such paintypically evokes escape or protective behaviors in animals or in a humanin order to remove itself, or protect itself, from further exposure tothe insult. However, inflammation, cellular and neuronal damage andother processes resulting from injury or disease can lead to states ofchronic pathological pain and enhanced sensitivity called hyperalgesia,in which the perception of pain is exaggerated. In such cases, there isa need for new compounds, new methods and new inventions that treat painand hyperalgesia.

In some cases, such as surgical procedures, the diminution orabolishment of nociceptive pain may be desired and the application ofanalgesia may be employed. A variety of compounds and medicines areknown to be effective in alleviating nociceptive pain and some areanalgesic such as opioids.

Post surgically, pain arising from the procedure, for example such aspain at or near the site of an incision, is common. In such cases, thereis a desire to alleviate pain and thus there is a need for newcompounds, new methods and new inventions that treat post-surgical andincisional pain.

The administration of opioids to treat pain is a well recognized andcommonly employed therapy in medicine. Unfortunately, tolerance toopioids (tachyphylaxis) and opioid-induced hyperalgesia can often resultduring the course of therapy. In such patients, increasingly higherdoses of opioids are needed to provide an acceptable level of painrelief. Administering these higher does, though, can cause adverse sideeffects implicating the safety concerns associated with opioids. Sideeffects associated with opioid administration may include respiratorydepression, constipation, nausea, and vomiting. Safety concerns includethe possibility of developing dependence, suffering withdrawal upondiscontinuation of treatment, and the potential for abuse. Tachyphylaxisis a phenomenon in which the repeated administration of a drug, such asa narcotic analgesic, results in a rapidly appearing and marked decreasein the effectiveness of that drug. In opioid-induced hyperalgesia,prolonged administration of opioids also results in a paradoxicalincrease in pain, or a hypersensitivity to a stimulus that is thought tobe unrelated to the original injury or insult. Opioid-inducedtachyphylaxis and opioid-induced hyperalgesia have been well documentedin animal models of nociception as well as in human clinical trials.These phenomena present significant clinical challenges for thetreatment of pain and therefore new compounds, new methods and newinventions to treat pain and/or to alleviate hyperalgesia and toleranceare needed.

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) arehighly reactive small molecules that include, for example, oxygen ionsand free radicals such as peroxide, hydroxyl radical and other speciessuch as peroxynitrite (OONO⁻). Peroxynitrite is the product of theinteraction between superoxide (O₂ ⁻) and nitric oxide (.NO). In cells,ROS and RNS form normally, as a by-product of normal metabolism, butthey also play an important role in the pathogenesis of many disorders,including those affecting the lung, the central nervous system andskeletal muscle. In addition, during times of stress (such as, forexample, hypoxia) ROS and/or RNS levels can increase significantly,which can lead to damage to cell components.

Peroxynitrite activity has been implicated in the development ofopiate-induced antinociceptive (pain) tolerance (tachyphylaxis) (Muscoliet al., 2007, J Clin Invest 117:3530-3539). In 2007, Muscoli proposedthat peroxynitrite formation is a key pathway in the development ofnarcotic analgesic tachyphylaxis. Previous attempts at reducing thedamaging effects of peroxynitrite have been focused on flavonoids andsimpler phenols, some of which have proven to be ineffective or toxicwhen administered to animals and man (Olmos et al., 2007, Med Res Rev27:1-64; Choi et al., 2002, Phytother Res 16:232-235). Thus,collectively there exists a need for new compounds, new methods and newinventions that decrease the level or activity of reactiveoxygen/nitrogen species, such as peroxynitrite.

Thus, there exists a need for compounds, compositions, and methods forpreserving or extending opioid analgesia without opioid dose-escalation.Similarly, there exists a need for compounds, compositions, and methodsthat are dose-sparing with respect to opioid therapy. The currentinvention fulfills these needs.

BRIEF SUMMARY OF INVENTION

The invention includes a compound according to Formula I or a saltthereof

wherein:

R¹ and R² are, independently, hydrogen or alkyl, or R¹ and R² formtogether, a radical according to the formula (CH₂)_(n), wherein 2≦n≦6;

R³ is, independently at each occurrence, hydrogen, alkyl, substitutedalkyl, alkenyl, alkynyl, halogen, phenyl, substituted phenyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, hydroxyl, alkoxy,cyano, nitro, acyl, carboxy, carboxyalkyl, or amido;

R⁴ is hydrogen, alkyl, substituted alkyl, or acyl; and

W is hydrogen, alkyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, carboxy, or carboxyalkyl.

In one embodiment, R¹ and R² are CH₃. In another embodiment, thecompound according to Formula I is selected from the group consisting of(S)-2-(2-hydroxyphenylamino)-thiazoline-5,5-dimethyl-4-carboxylic acid,(R)-2-(2-hydroxyphenylamino)-thiazoline-5,5-dimethyl-4-carboxylic acid,(R)-2-(2-Hydroxy-4-methoxyphenylamino)-thiazoline-4-carboxylic acid,(R)-2-(4-Fluoro-2-hydroxyphenylamino)-thiazoline-4-carboxylic acid,(R)-2-(3,5-Dichloro-2-hydroxy-4-methylphenylamino)-thiazoline-4-carboxylicacid, (R)-2-(5-tert-Butyl-2-hydroxyphenylamino)-thiazoline-4-carboxylicacid,(R)-2-(2-Hydroxy-4-methoxycarbonylphenylamino)-thiazoline-4-carboxylicacid,(R)-2-(5-Ethanesulfonyl-2-hydroxy-phenylamino)-thiazoline-4-carboxylicacid, (R)-2-(4-Chloro-2-hydroxyphenylamino)-thiazoline-4-carboxylicacid, (R)-2-(2-Hydroxy-5-methoxyphenylamino)-thiazoline-4-carboxylicacid,(S)-2-(2-Hydroxy-5-chlorophenylamino)-thiazoline-5,5-dimethyl-4-carboxylicacid methyl ester,(S)-2-(2-Hydroxy-5-chlorophenylamino)-thiazoline-5,5-dimethyl-4-carboxylicacid,(S)-2-(2-Hydroxy-5-methylphenylamino)-thiazoline-5,5-dimethyl-4-carboxylicacid methyl ester,(S)-2-(2-Hydroxy-5-methylphenylamino)-thiazoline-5,5-dimethyl-4-carboxylicacid,(S)-2-(2-Hydroxy-4-methoxyphenylamino)-thiazoline-5,5-dimethyl-4-carboxylicacid,(S)-2-(2-Hydroxy-4-methoxyphenylamino)-thiazoline-5,5-dimethyl-4-carboxylicacid methyl ester,(S)-2-(4-Chloro-2-hydroxyphenylamino)-thiazoline-5,5-dimethyl-4-carboxylicacid,(S)-2-(2-Hydroxy-5-nitrophenylamino)-thiazoline-5,5-dimethyl-4-carboxylicacid methyl ester,(S)-2-(2-Hydroxy-5-nitrophenylamino)-thiazoline-5,5-dimethyl-4-carboxylicacid,(S)-2-(5-Ethanesulfonyl-2-hydroxyphenylamino)-thiazoline-5,5-dimethyl-4-carboxylicacid methyl ester,(S)-2-(5-Ethanesulfonyl-2-hydroxyphenylamino)-thiazoline-5,5-dimethyl-4-carboxylicacid, (R)-2-(2-hydroxyphenylamino)-thiazoline-4-carboxylic acid methylester, (S)-2-(2-hydroxyphenylamino)-thiazoline-5,5-dimethyl-4-carboxylicacid methyl ester,(R)-2-(4-chloro-2-hydroxyphenylamino)-thiazoline-4-carboxylic acidmethyl ester,(S)-2-(4-chloro-2-hydroxyphenylamino)-thiazoline-5,5-dimethyl-4-carboxylicacid methyl ester,(R)-2-(4-chloro-2-hydroxyphenylamino)-thiazoline-4-carboxylic acid, and(R)-2-(2-hydroxyphenylamino)-thiazoline-5,5-dimethyl-4-carboxylic acidmethyl ester, mixtures thereof and salts thereof.

The invention also includes a method of making a compound according toFormula I, or a salt thereof. The method comprises: reacting a compoundaccording to formula A:

-   -   with a compound according to formula C:

-   -   to give a compound according to formula D:

The method further comprises deprotecting the compound according toformula D to give the compound according to Formula I or a salt thereof;

wherein

R¹ and R² are, independently, hydrogen or alkyl, or R¹ and R² formtogether a radical according to the formula (CH₂)_(n), wherein 2≦n≦6;

R³ is, independently at each occurrence, hydrogen, alkyl, substitutedalkyl, alkenyl, alkynyl, halogen, phenyl, substituted phenyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, hydroxyl, alkoxy,cyano, nitro, acyl, carboxy, carboxyalkyl, or amido;

R⁴ is hydrogen, alkyl, substituted alkyl, or acyl;

W is hydrogen, alkyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, carboxy, or carboxyalkyl;

Z is Cl, Br, or I; and

P is a thiol protecting group.

The invention further includes a method of making a compound accordingto Formula I. The method comprises reacting a compound according toformula A or a salt thereof:

-   -   with a compound according to formula B:

-   -   to give the compound according to Formula I or a salt thereof;        wherein

R¹ and R² are, independently, hydrogen or alkyl, or R¹ and R² formtogether a radical according to the formula (CH₂)_(n), wherein 2≦n≦6;

R³ is, independently at each occurrence, hydrogen, alkyl, substitutedalkyl, alkenyl, alkynyl, halogen, phenyl, substituted phenyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, hydroxyl, alkoxy,cyano, nitro, acyl, carboxy, carboxyalkyl, or amido;

R⁴ is hydrogen, alkyl, substituted alkyl, or acyl;

W is hydrogen, alkyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, carboxy, or carboxyalkyl; and

Z is Cl, Br, or I.

The invention further includes a method of diminishing the dosage of anarcotic analgesic required to achieve pain relief in a subject in needthereof, the method comprising the step of administering to the subjecta therapeutically effective amount of at least one compound according toFormula I or a salt thereof,

wherein:

R¹ and R² are, independently, hydrogen or alkyl, or R¹ and R² formtogether a radical according to the formula (CH₂)_(n), wherein 2≦n≦6;

R³ is, independently at each occurrence, hydrogen, alkyl, substitutedalkyl, alkenyl, alkynyl, halogen, phenyl, substituted phenyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, hydroxyl, alkoxy,cyano, nitro, acyl, carboxy, carboxyalkyl, or amido;

R⁴ is hydrogen, alkyl, substituted alkyl, or acyl; and

W is hydrogen, alkyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, carboxy, or carboxyalky.

whereby the dosage of the narcotic analgesic required to achieve painrelief in the subject

diminished.

In one embodiment, the compound is administered in a therapeutic dosingregimen or a preconditioning dosing regimen. In another embodiment, thesubject is a human.

The invention further includes a method of extending the duration ofaction or analgesic efficacy of a narcotic analgesic in a subject inneed thereof, the method comprising the step of administering to thesubject a therapeutically effective amount of at least one compoundaccording to Formula I or a salt thereof,

wherein:

R¹ and R² are, independently, hydrogen or alkyl, or R¹ and R² formtogether a radical according to the formula (CH₂)_(n), wherein 2≦n≦6;

R³ is, independently at each occurrence, hydrogen, alkyl, substitutedalkyl, alkenyl, alkynyl, halogen, phenyl, substituted phenyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, hydroxyl, alkoxy,cyano, nitro, acyl, carboxy, carboxyalkyl, or amido;

R⁴ is hydrogen, alkyl, substituted alkyl, or acyl; and

W is hydrogen, alkyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, carboxy, or carboxyalky.

whereby the duration of action or analgesic efficacy of the narcoticanalgesic in the subject is extended.

In one embodiment, the compound is administered in a therapeutic dosingregimen or a preconditioning dosing regimen. In another embodiment, thesubject is a human.

The invention further includes a method of preventing tachyphylaxis in asubject in need thereof, the method comprising the step of administeringto the subject a therapeutically effective amount of at least onecompound according to Formula I or a salt thereof,

wherein:

R¹ and R² are, independently, hydrogen or alkyl, or are together aradical according to the formula (CH₂)_(n), wherein 2≦n≦6;

R³ is, independently at each occurrence, hydrogen, alkyl, substitutedalkyl, alkenyl, alkynyl, halogen, phenyl, substituted phenyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, hydroxyl, alkoxy,cyano, nitro, acyl, carboxy, carboxyalkyl, or amido;

R⁴ is hydrogen, alkyl, substituted alkyl, or acyl;

W is hydrogen, alkyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, carboxy, or carboxyalkyl.

whereby tachyphylaxis in the subject is prevented.

In one embodiment, the compound is administered in a therapeutic dosingregimen or a preconditioning dosing regimen. In another embodiment, thesubject is a human.

The invention further includes a method of reducing the level ofactivity of reactive oxygen species and reactive nitrogen species in asubject in need thereof, the method comprising the step of administeringto the subject a therapeutically effective amount of at least onecompound according to Formula I or a salt thereof,

wherein:

R¹ and R² are, independently, hydrogen or alkyl, or R¹ and R² formtogether a radical according to the formula (CH₂)_(n), wherein 2≦n≦6;

R³ is, independently at each occurrence, hydrogen, alkyl, substitutedalkyl, alkenyl, alkynyl, halogen, phenyl, substituted phenyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, hydroxyl, alkoxy,cyano, nitro, acyl, carboxy, carboxyalkyl, or amido;

R⁴ is hydrogen, alkyl, substituted alkyl, or acyl; and

W is hydrogen, alkyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, carboxy, or carboxyalkyl.

whereby the level of activity of reactive oxygen species and reactivenitrogen species in the subject is reduced.

In one embodiment, the reactive oxygen species is peroxynitrite. Inanother embodiment, the subject exhibits symptoms of at least onedisorder from the group consisting of: stroke, myocardial infarction,chronic heart failure, circulatory shock, chronic inflammatory diseases,cancer, neurogenerative disorders, sleep apnea, diabetes, narcoticanalgesic tachyphylaxis, pancreatitis pain, addictive behavior, andhypertension. Yet in another embodiment, the compound is administered ina therapeutic dosing regimen or a preconditioning dosing regimen. Yet inanother embodiment, the level of activity of reactive oxygen species isreduced by diminishing reactive oxygen species production. Yet inanother embodiment, the subject is a human.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description of preferred embodiments of theinvention, will be better understood when read in conjunction with theappended drawings. For the purpose of illustrating the invention, thereare shown in the drawings embodiments which are presently preferred. Itshould be understood, however, that the invention is not limited to theprecise arrangements and instrumentalities of the embodiments shown inthe drawings. In the drawings:

FIG. 1 depicts the results of an example experiment demonstrating thatcompound 2 dose-dependently preserves fentanyl analgesia in rat tailflick model.

FIG. 2 depicts the results of an example experiment demonstrating thatcompound 2 dose-dependently preserves fentanyl analgesia in Hargreavesthermal hyperalgesia model.

FIG. 3 depicts the results of an example experiment demonstrating thatcompound 2 preserves fentanyl analgesia, allowing smaller doses offentanyl to elicit full or significant analgesia in rat as measured byHargreaves thermal endpoints. Vehicle or Compound 2 (25 mg/kg, IV) weregiven 15 min before fentanyl. Multiples doses of fentanyl given to bothgroups at times indicated.

FIG. 4 depicts the results of an example experiment demonstrating that asingle dose of compound 2 preserves fentanyl analgesia and blockshyperalgesia even after three doses of fentanyl.

FIG. 5 depicts the results of an example experiment demonstrating that asingle dose of compound 2 preserves fentanyl analgesia and inhibitsdevelopment of hyperalgesia even after four doses of fentanyl.

FIG. 6 depicts the results of an example experiment demonstrating thatcompound 2 preserves fentanyl analgesia by reducing tolerance andinhibiting hyperalgesia.

FIG. 7 depicts the results of an example experiment demonstrating thatcompound 2 attenuates established opioid-induced tolerance/hyperalgesia.

FIG. 8 depicts the results of an example experiment demonstrating thatcompound 2 inhibits capsaicin-mediated hyperalgesia.

FIG. 9, comprising FIG. 9A and FIG. 9B, depicts the results of anexample experiment demonstrating that compound 2 inhibits thedevelopment of hyperalgesia following incisional surgery.

FIG. 10, comprising FIG. 10A and FIG. 10B, depicts the results of anexample experiment demonstrating that compound 2 reverses hyperalgesiafollowing incisional surgery.

FIG. 11 depicts the results of an example experiment demonstrating thatcompound 2 inhibits hyperalgesia caused by SIN-1, a known producer ofperoxynitrite.

FIG. 12 is an illustration of the structure of(S)-2-(2-hydroxyphenylamino)-thiazoline-5,5-dimethyl-4-carboxylic acidhydrochloride (compound 1) as determined by crystallographic X-raydiffraction pattern analysis.

FIG. 13 is an illustration of the structure of(S)-2-(5-ethanesulfonyl-2-hydroxyphenylamino)-thiazoline-5,5-dimethyl-4-carboxylicacid methyl ester (compound 32) as determined by crystallographic X-raydiffraction pattern analysis.

FIG. 14 is an illustration of the structure of(R)-2-(4-fluoro-2-hydroxyphenylamino)-thiazoline-4-carboxylic acid(compound 6) as determined by crystallographic X-ray diffraction patternanalysis.

FIG. 15 is an illustration of the crystal packing structure of(S)-2-(5-ethanesulfonyl-2-hydroxyphenylamino)-thiazoline-5,5-dimethyl-4-carboxylicacid methyl ester (compound 32) as determined by crystallographic X-raydiffraction pattern analysis.

FIG. 16 is a graph illustrating the effect of a single preemptive doseof Compound 2, 25 mg/kg i.v., on fentanyl induced toxicity (6 hourlydoses of 25 μg/kg i.v. fentanyl).

FIG. 17 is a graph illustrating the effect of preemptively dosedCompound 2, 25 mg/kg i.v., on fentanyl analgesia (6 hourly doses of 25μg/kg i.v. fentanyl).

FIG. 18, comprising FIG. 18A and FIG. 18B, illustrates the effect ofpreemptively dosed 25 mg/kg Compound 2 i.v. on FCA induced thermalhyperalgesia and tactile allodynia.

FIG. 19, comprising FIG. 19A and FIG. 19B, illustrates the effect of3-100 mg/kg Compound 2 p.o. on FCA tactile allodynia when dosed 24-72hours post-FCA

FIG. 20, comprising FIG. 20A and FIG. 20B, illustrates the effect of 10and 30 mg/kg Compound 2 p.o. on FCA induced edema when dosed eitherpreemptively or 24 hours post-FCA.

FIG. 21, comprising FIG. 21A and FIG. 21B, illustrates the effect of 10and 30 mg/kg Compound 2 p.o. on carrageenan-induced thermal hyperalgesiaand edema when dosed curatively.

FIG. 22, comprising FIG. 22A and FIG. 22B, illustrates the effect of 10and 30 mg/kg Compound 2 p.o. on carrageenan-induced thermal hyperalgesiaand edema when dosed preemptively.

FIG. 23 is a graph illustrating the effect of 10 or 50 mg/kg Compound 2p.o. on spinal nerve ligation induced tactile allodynia.

FIG. 24 is a graph illustrating the effect of 50 mg/kg Compound 2 p.o.on spinal nerve ligation induced tactile allodynia.

FIG. 25 is a graph illustrating the effect of dosing of single dose(day 1) 30 mg/kg Compound 2 p.o. on spinal nerve ligation inducedtactile allodynia.

FIG. 26 is a graph illustrating the effect of repeated QD dosing (days1-5) of 30 mg/kg Compound 2 p.o. on spinal nerve ligation inducedtactile allodynia.

FIG. 27 is a bar graph illustrating the effect of 30, 100 and 300 mg/kgCompound 2 p.o. on latency to fall in the accelerating rotarod assay ofataxia.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to compounds, compositions, and methods fordiminishing pain in a subject in need thereof. In one embodiment, theinvention is a compound according to Formula I:

In another embodiment, the invention relates to a composition comprisinga compound according to formula I. In another embodiment, the inventionis a method of administering a compound and/or composition of theinvention to provide analgesia. In various embodiments, the inventionrelates to compounds, compositions, and methods for reducing orabolishing incisional pain in a subject in need thereof. The inventionalso provides methods for reducing or abolishing hyperalgesia in asubject in need thereof. It is also an aspect of the invention that thecompounds and/or compositions disclosed herein can be administered to asubject to reduce, prevent or reverse hyperalgesia occurring in responseto the administration of a drug, such as a narcotic analgesic.

In one aspect, the invention is a compound and/or composition that leadsto an increase in the duration of action of narcotic analgesic agentssuch as, for example, fentanyl or morphine. In another aspect, theinvention is a method that leads to an increase in the duration ofaction of narcotic analgesic agents, such as fentanyl or morphine.

In another embodiment, the invention is a compound and/or compositionthat diminishes the effective dosage of a narcotic analgesic agent suchas, for example, fentanyl or morphine. In still another embodiment, theinvention is a method that leads to a decrease in the effective dosageof a narcotic analgesic agent, such as fentanyl or morphine. A decreasein the effective dosage means that, when administered in conjunctionwith the compounds, compositions, or methods of the invention, a lowerdosage of a narcotic analgesic is sufficient to achieve the same levelof analgesia as compared with when the analgesic agent is administeredalone.

Definitions:

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are described.

As used herein, each of the following terms has the meaning associatedwith it in this section.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

The term “about” will be understood by persons of ordinary skill in theart and will vary to some extent on the context in which it is used.

As used herein, the term “modulate” is meant to refer to any change inbiological state, i.e. increasing, decreasing, and the like.

As used herein, a “therapeutically effective amount” is the amount of atherapeutic composition sufficient to provide a beneficial effect to asubject to which the composition is administered.

The terms “inhibiting,” “reducing,” “preventing,” or “diminishing,” andvariations of these terms, as used herein include any measurabledecrease, including complete or substantially complete inhibition.

The term “or,” as used herein, means “and/or” unless explicitlyindicated to refer to alternatives only or the alternatives are mutuallyexclusive, although the disclosure supports a definition that refers toonly alternatives and “and/or.”

The term “treat” or “treatment,” as used herein, refers to thealleviation (i.e., “diminution”) and/or the elimination of asign/symptom or a source of a sign/symptom. By way of severalnon-limiting examples, a disorder can be treated by alleviating asign/symptom of a disorder. A sign/symptom of a disorder can also betreated by altogether eliminating a sign/symptom of the disorder.

The phrase “therapeutic dosing regimen” as used herein, means toadminister a compound of the invention to a subject, at time when thesubject is experiencing a disorder as elsewhere described herein, in amanner, including dosage, timing of doses, and frequency of doses, so asto produce a beneficial consequence in the subject, such as treatment ofa disorder, or a symptom of a disorder, in the subject.

The phrase “preconditioning dosing regimen” as used herein, means toadminister a compound of the invention to a subject, at a time beforethe subject is experiencing a disorder as elsewhere described herein, ina manner, including dosage, timing of doses, and frequency of doses, soas to produce a beneficial consequence in the subject, such as treatmentof a disorder, or a symptom of a disorder, in the subject.

The term “subject,” as used herein, refers to a mammal, including dogs,cats, pigs, cows, sheep, goats, horses, rats, and mice and humans.Preferably the subject is a human.

As used herein, a “decrease in the effective dosage” means that, whenadministered in conjunction with the compositions or methods of theinvention, a lower overall dosage of a narcotic analgesic, over time, issufficient to achieve the same level of analgesia as compared with whenthe analgesic agent is administered alone.

Description

Tachyphylaxis is a phenomenon in which the repeated administration of adrug, such as a narcotic analgesic, results in a rapidly appearing andmarked decrease in the effectiveness of that drug. It is a furtheraspect of the invention that the compounds and/or compositions describedherein can be administered to a subject to reduce, prevent or reversetachyphylaxis occurring in response to the administration of a drug,such as a narcotic analgesic.

Narcotic analgesics are known to cause respiratory depression. Thecompounds, compositions, and methods of the invention can be used toprevent a loss of normal breathing, or to restore normal breathing aftera loss occurs. One non-limiting example of a loss of normal breathing isrespiratory depression. Respiratory depression results inhypoventilation, which further results in hypoxia. A primary initialclinical manifestation of hypoxia is drowsiness or excessive daytimesleepiness. Accordingly, drugs that cause decreased respiratory driveand the resulting hypoxia are sometimes limited in their usefulness dueto the fear of a life-threatening respiratory depression and/or theexcessive daytime sleepiness that negatively impacts quality of life.

There are a wide variety of disorders that have loss of normal breathingor respiratory depression as a primary or secondary feature of thedisorder, which can be treated using the compounds, compositions, andmethods of the present invention. Examples of a primary loss of normalbreathing include: apneas (central, mixed and obstructive) andcongenital central hypoventilation syndrome. Secondary loss of normalbreathing may be due to certain drugs (e.g., anesthetics, sedatives,anxiolytics, hypnotics, alcohol, opioid analgesics), chroniccardio-pulmonary diseases (e.g., heart failure, chronic bronchitis,emphysema, and impending respiratory failure), excessive weight (e.g.,obesity-hypoventilation syndrome) and/or factors that affect theneurological system (e.g., stroke, tumor, trauma, radiation damage,ALS).

Generally, patients in need of analgesia or anesthesia may receive oneagent, or a combination of multiple agents, to create a state of partialor full unconsciousness to allow for medical procedures, such assurgery, to be performed. A common undesirable action of many agentsused for analgesia and anesthesia (e.g., opioid analgesics,barbiturates, benzodiazepines, inhaled anesthetics, propofol) isrespiratory depression. Examples of opioid analgesics include morphine,codeine, fentanyl, buprenorphine, meperidine, methadone, sufentanil,alfentanil, and the like. Examples of barbiturates include allobarbital,alphenal, amobarbital, aprobarbital, barbexaclone, barbital,brallobarbital, butabarbital, butalbital, butobarbital, butallylonal,crotylbarbital, cyclobarbital, cyclopal, ethallobarbital, febarbamate,heptabarbital, hexethal, hexobarbital, mephobarbital, metharbital,methohexital, methylphenobarbital, narcobarbital, nealbarbital,pentobarbital, phenobarbital, probarbital, propallylonal, proxibarbal,proxibarbital, reposal, secbutabarbital, secobarbital, sigmodal,talbutal, thialbarbital, thiamylal, thiobarbital, thiobutabarbital,thiopental, valofane, vinbarbital, and vinylbital. Examples ofbenzodiazepines include midazolam, clonazepam, diazepam, alprazolam andthe like. Examples of inhaled anesthetics included halothane, enflurane,isoflurane, sevoflurane, desflurane, and the like. Not only can therespiratory depressant effect occur soon after administration of theagent, but the effects of the anesthetic and/or analgesic agent canlinger for hours or days after the procedure. The compounds,compositions, and methods of the invention can be used to diminish,prevent or reverse drug-induced respiratory depression.

In one embodiment of the invention the compounds and/or compositionsdisclosed herein diminish the level of, or inhibit the activity of, ROSor RNS (such as, by way of example only, peroxynitrite). In anotherembodiment, the invention includes a compound and/or composition thatdiminishes the level or inhibits the activity of ROS and RNS, such asperoxynitrite, which leads to an increase in the duration of action of anarcotic analgesic agent, such as, by way of non-limiting examples,fentanyl and morphine. In another embodiment, the invention includes acompound and/or composition that diminishes the level or inhibits theactivity of ROS and RNS, such as, by way of non-limiting example,peroxynitrite, which contributes to a decrease in the effective dosageof a narcotic analgesic agent such as, by way of non-limiting examples,fentanyl and morphine. In still another embodiment, the invention is amethod of diminishing the level or activity of ROS and RNS, such as, byway of non-limiting example, peroxynitrite, which leads to a decrease inthe effective dosage of a narcotic analgesic agent, such as, by way ofnon-limiting examples, fentanyl or morphine.

The therapeutic uses of the compounds, compositions, and methods of theinvention include, but are not limited to, combating the effects of ROSand RNS, such as, by way of non-limiting example, peroxynitrite.Potential disease targets are extensive and include, but are not limitedto; stroke, myocardial infarction, chronic heart failure, circulatoryshock, chronic inflammatory diseases, cancer, neurogenerative disorders,sleep apnea, diabetes, narcotic analgesic tachyphylaxis, pancreatitispain, addictive behavior, and hypertension (see also Pacher 2007,Physiol Rev 87:315-424).

While not wishing to be limited by any specific example, several uses ofthe compounds, compositions, and methods of the invention include:extending the therapeutic potency and efficacy of narcotic analgesicsfor pain relief, reducing the side effects of naracotic analgesics, andimproving the safety profile of narcotic, and other, analgesics,improving muscle strength in patients experiencing hypoxia (see Clanton,2007, J Appl Physiol 102:2379-2388) and restoring the ability ofbreathing control chemoreceptors to function properly (Zakynthinos etal., 2007, Am J Respir Crit Care Med 175:62-68). Additional examplesalso include: enhancing muscle strength for certain types ofneuromuscular disease such as Duchenne Muscular Dystrophy, (Williams etal., 2007, Am J Physiol Heart Circ Physiol 293:H1969-H1977; Tidball andWehling-Hendricks, 2007, J Appl Physiol 102:1677-1686; Whitehead et al.,2008, J Physiol 7:2003-2014), Huntington's disease (Perez-De La Cruz etal., 2009, Behav Brain Res 199:210-217), and Parkinson's Disease (Pinnenet al., 2009, J Med Chem 52:559-563).

In one nonlimiting embodiment, the compounds, compositions, and methodsof the invention are used as an anti-oxidant therapy, including forexample, anti-oxidant therapy to treat psychiatric disorders, metabolicdisorders, and addiction. Anti-oxidant therapy has been suggested for awide variety of psychiatric and metabolic disorders (Bhardwaj et al.,2009, Gastroenterology 136:149-159; Ng et al., 2008, Int JNeuropsychopharmacol 11:851-76). For example, N-acetylcysteine, anantioxidant, has also been proposed for addiction behavior/neurologicaldamage including methamphetamine (Imam et al., 2001, Ann NY Acad Sci939:366-380), cocaine or heroin addiction (Madayag et al., 2007, JNeuroscience 27:13968-13976; LaRowe et al., 2007, Am J Psychiatry164:1115-1117; Zhou and Kalicas, 2008, Biol Psychiatry 63:338-340;Mardikian et al., 2007, Prog Neuro-psychopharmacol Biol Psychiatry31:389-394).

In another aspect of the invention, the analgesic pain relieving effectsof narcotic analgesics are preserved and decoupled from their dangerousand discomforting side effects. For example, for treatment of moderateto severe pain, the narcotic analgesics (e.g., morphine) are still themainstay of therapy for many indications. The major drawbacks to the useof narcotic analgesics include a variety of side effects includingrespiratory depression, constipation, sedation and urinary retention.Thus, in one embodiment, the compounds, compositions, and methods of theinvention extend the analgesic effect of narcotic analgesics. In anotherembodiment, the compound, compositions, and methods of the inventionblunt the respiratory depression associated with the use of narcoticanalgesics. In still another embodiment, the compound, compositions, andmethods of the invention extend the analgesic effect of narcoticanalgesics and blunt the respiratory depression associated with the useof narcotic analgesics. In various embodiments of the invention, thedecreased dose of narcotic analgesic necessary to achieve the same levelof analgesia provides for fewer or less severe side effects, such as,respiratory depression, constipation, sedation, and urinary retention.Similarly, reducing the dose of narcotic needed to achieve adequate painrelief can also reduce the likelihood of a patient developing dependenceon opioids or suffering opioid withdrawal upon discontinuation oftreatment, thereby reducing the likelihood of narcotic abuse.

Methods of treatment of the invention also include administration of acompound and/or composition of the invention to stabilize the breathingrhythm of a subject, as well as administration of a compound and/orcomposition of the invention to increase minute ventilation of asubject. In an embodiment, a method of the invention includesadministration of a compound and/or composition of the invention tostabilize the breathing rhythm of a subject. In another embodiment, amethod of the invention includes administration of a compound and/orcomposition of the invention to increase minute ventilation of asubject. In an aspect, minute ventilation of a subject is increased atthe level of the brainstem respiratory control center in the nucleustractus solitarius.

Compounds of the Invention

The compounds of the invention include those compounds having astructure according to Formula I or a salt thereof.

In Formula I, R¹ and R² can be, independently, hydrogen or alkyl.Alternatively, R¹ and R² can together form a radical according to theformula (CH₂)_(n), wherein 2≦n≦6.

R³ can be, independently at each occurrence, hydrogen, alkyl,substituted alkyl, alkenyl, alkynyl, halogen, phenyl, substitutedphenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl,hydroxyl, alkoxy, cyano, nitro, acyl, carboxy, carboxyalkyl, or amido.

R⁴ can be hydrogen, alkyl, substituted alkyl, or acyl.

W can be hydrogen, alkyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, carboxy, or carboxyalkyl.

As used herein, the term “alkyl”, by itself or as part of anothersubstituent means a saturated straight, branched, or cyclic hydrocarbonhaving the number of carbon atoms designated according to the notationC_(x)-C_(y) wherein 1≦x≦y≦10. According to this notation, (C₁-C₆)alkylmeans the group of saturated straight, branched, or cyclic hydrocarbonshaving from 1 to 6 carbon atoms. Examples of alkyl groups include, butare not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl,tertbutyl, pentyl, neopentyl, hexyl, cyclohexyl and cyclopropylmethyl.

As used herein the term “acyl” refers to an alkyl group connected to acarbonyl group. Acyl groups may be represented by the formula—C(O)alkyl. Examples of acyl groups include, but are not limited to,acetyl, pivaloyl, propanoyl, and isobutanoyl.

The term “alkenyl” employed alone or in combination with other terms,means, unless otherwise stated, a stable monounsaturated ordi-unsaturated straight chain, branched chain or cyclic hydrocarbongroup having the number of carbon atoms designated according to thenotation C_(x)-C_(y) wherein 1≦x≦y≦10. According to this notation,(C₁-C₆)alkenyl means the group of mono- or di-unsaturated straight,branched, or cyclic hydrocarbons having from 1 to 6 carbon atoms.Examples of alkenyl radicals include, but are not limited to, vinyl,propenyl (allyl), crotyl, isopentenyl, butadienyl, 1,3-pentadienyl,1,4-pentadienyl, cyclopentenyl, cyclopentadienyl and the higher homologsand isomers. Alkenyl groups may be represented by according to thenotation CH₂═CHCH₂—.

The term “alkoxy” employed alone or in combination with other termsrefers to a group having the structure “alkyl-O—”. Examples include butare not limited to, methoxy (CH₃O—), ethoxy (CH₃CH₂O—), 1-propoxy(CH₃CH₂CH₂O—), and isopropoxy (CH₃(CHO—)CH₃). Preferred embodimentsinclude (C₁-C₃)alkoxy, particularly ethoxy and methoxy.

The term “alkoxycarbonyl,” as used herein, refers to an alkoxy group,bound to a carbonyl group, said alkoxycarbonyl group having thestructure “alkoxy-C(O)—”

The term “carboxy” or “carboxyl,” as used herein, refers to a —CO₂Hgroup.

The term “carboxyalkyl,” as used herein, refers to a compound having theformula “—C(O)O-alkyl”.

The term “aryl”, employed alone or in combination with other terms,means a carbocyclic aromatic system containing one or more rings(typically one, two or three rings) wherein said rings may be attachedtogether in a pendent manner, such as a biphenyl, or may be fused, suchas in naphthalene. Examples include, but are not limited to, phenyl,anthracyl, and naphthyl.

The term “heterocycle” or “heterocyclic,” as defined herein, refers tomonocyclic, bicyclic, or tricyclic ring systems containing at least oneheteroatom. Monocyclic ring systems are exemplified by any 3- or4-membered ring containing a heteroatom independently selected fromoxygen, nitrogen and sulfur; or a 5-, 6- or 7-membered ring containingone, two, three, or four heteroatoms wherein the heteroatoms areindependently selected from nitrogen, oxygen and sulfur. The 5-memberedring has from 0-2 double bonds and the 6- and 7-membered ring has from0-3 double bonds. Representative examples of monocyclic ring systemsinclude, but are not limited to, azetidinyl, azepinyl, aziridinyl,diazepinyl, 1,3-dioxolanyl, dioxanyl, dithianyl, furyl, imidazolyl,imidazolinyl, imidazolidinyl, isothiazolyl, isothiazolinyl,isothiazolidinyl, isoxazolyl, isoxazolinyl, isoxazolidinyl,N-methylpiperazinyl, morpholinyl, oxadiazolyl, oxadiazolinyl,oxadiazolidinyl, oxazolyl, oxazolinyl, oxazolidinyl, piperazinyl,piperidinyl, pyranyl, pyrazinyl, pyrazolyl, pyrazolinyl, pyrazolidinyl,pyridyl, pyrimidinyl, pyridazinyl, pyrrolyl, pyrrolinyl, pyrrolidinyl,tetrahydrofuranyl, tetrahydrothienyl, tetrazinyl, tetrazolyl,1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,3,4-thiadiazolyl,thiadiazolinyl, thiadiazolidinyl, thiazolyl, thiazolinyl, thiazolidinyl,thienyl, thiomorpholinyl, 1,1-dioxidothiomorpholinyl, thiopyranyl,triazinyl, triazolyl, and trithianyl.

The term “heteroaryl” refers specifically to mono and polycyclicheterocycles having aromatic character. Examples of monocyclicheteroaryl groups include, but are not limited to, pyridyl, pyrazinyl,pyrimidinyl, pyridazinyl, thienyl, furyl, pyrrolyl, imidazolyl,thiazolyl, oxazolyl, pyrazolyl, isothiazolyl, 1,2,3-triazolyl,1,2,4-triazolyl, 1,3,4-triazolyl, tetrazolyl, 1,2,3-thiadiazolyl,1,2,3-oxadiazolyl, 1,3,4-thiadiazolyl and 1,3,4-oxadiazolyl.

Examples of polycyclic heteroaryl groups include, but are not limitedto, indolyl, indolinyl, quinolyl, tetrahydroquinolyl, isoquinolyl,1,2,3,4-tetrahydroisoquinolyl, cinnolinyl, quinoxalinyl, quinazolinyl,phthalazinyl, 1,8-naphthyridinyl, 1,4-benzodioxanyl, coumarin,dihydrocoumarin, benzofuryl, 2,3-dihydrobenzofuryl, 1,2-benzisoxazolyl,benzothienyl, benzoxazolyl, benzthiazolyl, purinyl, benzimidazolyl,benztriazolyl, thioxanthinyl, carbazolyl, carbolinyl, acridinyl,pyrrolizidinyl, and quinolizidinyl.

The term “halogen” means, unless otherwise stated, a fluorine, chlorine,bromine, or iodine atom.

The term “(C_(x)-C_(y))perfluoroalkyl,” wherein x<y, means an alkylgroup with a minimum of x carbon atoms and a maximum of y carbon atoms,wherein all hydrogen atoms are replaced by fluorine atoms. Examplesinclude, but are not limited to, trifluoromethyl and pentafluoroethyl.

Tautomerism

“Tautomers” are structurally distinct isomers that interconvert bytautomerization. “Tautomerization” is a form of isomerization involvingthe migration of a proton accompanied by changes in bond order, oftenthe interchange of a single bond with an adjacent double bond. Wheretautomerization is possible, (e.g., in solution), a chemical equilibriumof tautomers can be reached. One well known example of tautomerizationis between a ketone and its corresponding enol. Compounds according toFormula I may also undergo tautomerization and may exist in Form 1, Form2, or as a mixture thereof.

For purposes of the present invention, all compounds will be drawn inonly one tautomeric configuration. It should be understood, though, thatboth tautomeric forms of a given compound are contemplated and withinthe scope of the present invention. Thus, any discussion of any compounddisclosed herein should be understood to include both tautomeric formsof that compound, unless otherwise specified.

Acid Addition Salts

Compounds according to Formula I contain a basic nitrogen that can beprotonated by a sufficiently strong protic acid. Although anysufficiently strong protic acid may be used, pharmaceutically-acceptableacids are preferred so that pharmaceutically-acceptable acid additionsalts are formed when pharmaceutical compositions are desired.Pharmaceutically-acceptable acids refers to those acids that are nottoxic or otherwise biologically undesirable. Pharmaceutically acceptableacid addition salts may be formed with pharmaceutically-acceptableinorganic acids including, but not limited to, hydrochloric acid,hydrobromic acid, sulfuric acid, sulfamic acid, nitric acid, phosphoricacid, and the like.

Pharmaceutically acceptable acid addition salts may also be formed withpharmaceutically acceptable organic acids. Examples ofpharmaceutically-acceptable organic acids, include but are not limitedto, acetic acid, trifluoroacetic acid, adipic acid, ascorbic acid,aspartic acid, benzenesulfonic acid, benzoic acid, butyric acid,camphoric acid, camphorsulfonic acid, cinnamic acid, citric acid,digluconic acid, ethanesulfonic acid, glutamic acid, glycolic acid,glycerophosphoric acid, hemisulfic acid, hexanoic acid, formic acid,fumaric acid, 2-hydroxyethanesulfonic acid (isethionic acid), lacticacid, hydroxymaleic acid, malic acid, malonic acid, mandelic acid,mesitylenesulfonic acid, methanesulfonic acid, naphthalenesulfonic acid,nicotinic acid, 2-naphthalenesulfonic acid, oxalic acid, pamoic acid,pectinic acid, phenylacetic acid, 3-phenylpropionic acid, pivalic acid,propionic acid, pyruvic acid, salicylic acid, stearic acid, succinicacid, sulfanilic acid, tartaric acid, p-toluenesulfonic acid, undecanoicacid, and the like.

Methods of Preparation

Compounds of the invention or a salt thereof may be prepared accordingto the following general schemes. For example, according to Scheme 1, acompound of the invention may be prepared by reacting a compoundaccording to formula A, wherein Z is a leaving group, with anaminoalkylthiol B. This results in product compounds according toFormula I.

Alternatively, a protected aminoalkylthiol C can be reacted with acompound of formula A, wherein Z is a leaving group and P is a thiolprotecting group, to give the corresponding adduct D. SubsequentS-deprotection produces compounds according to Formula I. Suitable thiolprotecting groups are identified in Wuts, et al. Greene's ProtectiveGroups in Organic Synthesis, 4^(th) Edition. New York. John Wiley &Sons. 2007, the entirety of which is incorporated herein by reference.Preferred thiol protecting groups include triphenylmethyl, also referredto as “trityl” or “Tr”, and 4-methoxybenzyl.

In certain embodiments, compounds according to formula A may be preparedbeginning with a substituted phenol, according to formula E, as is shownin Scheme 3. Depending upon the nature of R³, a compound according toformula E may be commercially available or easily prepared according toknown methods in organic chemistry.

In Scheme 3, substituted phenol E can be nitrated to give thecorresponding ortho-nitrophenol F. This material can then be reduced toprovide the ortho-aminophenol G. The aminophenol G can subsequently bereacted with potassium ethyl xanthate (potassium(carbodithiolatooxy)ethane) to produce the cyclized2-mercaptobenzoxazole H. Reaction of the 2-mercaptobenzoxazole H with achlorinating agent, such as thionyl chloride, affords the desired2-chlorobenzoxazole J.

In certain embodiments, the nitration and/or reduction steps may beavoided by using a commercially available compound corresponding toadvanced intermediate F or G.

Pharmaceutical Compositions and Therapies

The invention also encompasses pharmaceutical compositions(“compositions”) and methods of their use. These pharmaceuticalcompositions may comprise an “active ingredient” (one or more compoundsof the invention, or pharmaceutically acceptable salts thereof) incombination with one or more pharmaceutically acceptable agents. Thecompositions set forth herein can be used alone or in combination withadditional compounds to produce additive, complementary, or synergisticeffects.

In an embodiment, the pharmaceutical compositions useful for practicingthe invention may be administered to deliver a dose of between 1ng/kg/day and 100 mg/kg/day. In another embodiment, the pharmaceuticalcompositions useful for practicing the invention may be administered todeliver a dose of between 1 ng/kg/day and 500 mg/kg/day.

In certain embodiments, the compositions of the invention may includeone or more pharmaceutically acceptable carriers. Pharmaceuticallyacceptable carriers, which are useful, include, but are not limited to,glycerol, water, saline, ethanol and other pharmaceutically acceptablesalt solutions such as phosphates and salts of organic acids. Examplesof these and other pharmaceutically acceptable carriers are described inRemington's Pharmaceutical Sciences (1991, Mack Publication Co., NewJersey).

The pharmaceutical compositions may be prepared, packaged, or sold inthe form of a sterile injectable aqueous or oily suspension or solution.This suspension or solution may be formulated according to the knownart, and may comprise, in addition to the active ingredient, additionalingredients such as anti-oxidants, dispersing agents, wetting agents, orsuspending agents described herein. Such sterile injectable formulationsmay be prepared using a non-toxic parenterally-acceptable diluent orsolvent, such as water or 1,3-butane diol, for example. Other acceptablediluents and solvents include, but are not limited to, Ringer'ssolution, isotonic sodium chloride solution, and fixed oils such assynthetic mono- or di-glycerides.

Pharmaceutical compositions that are useful in the methods of theinvention may be administered, prepared, packaged, and/or sold informulations suitable for intravenous, subcutaneous, sublingual, oral,rectal, vaginal, parenteral, topical, pulmonary, intranasal, buccal,ophthalmic, or another route of administration. Other contemplatedformulations include projected nanoparticles, liposomal preparations,resealed erythrocytes containing the active ingredient, andimmunologically-based formulations.

The compositions of the invention may be administered via numerousroutes, including, but not limited to, intravenous, subcutaneous,sublingual, oral, rectal, vaginal, parenteral, topical, pulmonary,intranasal, buccal, or ophthalmic administration routes. The route(s) ofadministration will be readily apparent to the skilled artisan and willdepend upon any number of factors including the type and severity of thedisorder being treated, the type and age of the veterinary or humanpatient being treated, and the like.

Pharmaceutical compositions that are useful in the methods of theinvention may be administered systemically in intravenous andsubcutaneous liquid formulations, oral and sublingual solidformulations, ophthalmic, suppository, aerosol, topical or other similarformulations. In addition to the compound such as heparin sulfate, or abiological equivalent thereof, such pharmaceutical compositions maycontain pharmaceutically-acceptable carriers and other ingredients knownto enhance and facilitate drug administration. Other possibleformulations, such as nanoparticles, liposomes, resealed erythrocytes,and immunologically based systems may also be used to administercompounds according to the methods of the invention.

Compounds which are identified using any of the methods describedherein, and combinations of such compounds, may be formulated andadministered to a subject for treatment of disordered control ofbreathing.

Such a pharmaceutical composition may consist of the active ingredientalone, in a form suitable for administration to a subject, or thepharmaceutical composition may comprise at least one active ingredientand one or more pharmaceutically acceptable carriers, one or moreadditional ingredients, or some combination of these. As describedelsewhere, the active ingredient may be present in the pharmaceuticalcomposition in the form of a physiologically or pharmaceuticallyacceptable salt, such as in combination with a physiologically orpharmaceutically acceptable cation or anion, as is well known in theart.

An obstacle for topical administration of pharmaceuticals is the stratumcorneum layer of the epidermis. The stratum corneum is a highlyresistant layer comprised of protein, cholesterol, sphingolipids, freefatty acids and various other lipids, and includes cornified and livingcells. One of the factors that limit the penetration rate (flux) of acompound through the stratum corneum is the amount of the activesubstance that can be loaded or applied onto the skin surface. Thegreater the amount of active substance which is applied per unit of areaof the skin, the greater the concentration gradient between the skinsurface and the lower layers of the skin, and in turn the greater thediffusion force of the active substance through the skin. Therefore, aformulation containing a greater concentration of the active substanceis more likely to result in penetration of the active substance throughthe skin, and more of it, and at a more consistent rate, than aformulation having a lesser concentration, all other things being equal.

The formulations of the pharmaceutical compositions described herein maybe prepared by any method known or hereafter developed in the art ofpharmacology. In general, such preparatory methods include the step ofbringing the active ingredient into association with a carrier or one ormore other accessory ingredients, and then, if necessary or desirable,shaping or packaging the product into a desired single- or multi-doseunit.

Although the descriptions of pharmaceutical compositions provided hereinare principally directed to pharmaceutical compositions which aresuitable for ethical administration to humans, it will be understood bythe skilled artisan that such compositions are generally suitable foradministration to subjects of all sorts.

Modification of pharmaceutical compositions suitable for administrationto humans in order to render the compositions suitable foradministration to various animals is well understood, and the ordinarilyskilled veterinary pharmacologist can design and perform suchmodification with merely ordinary, if any, experimentation. Subjects towhich administration of the pharmaceutical compositions of the inventionis contemplated include, but are not limited to, humans and otherprimates, mammals including commercially relevant mammals such ascattle, pigs, horses, sheep, cats, and dogs.

Pharmaceutical compositions that are useful in the methods of theinvention may be prepared, packaged, or sold in formulations suitablefor intravenous, subcutaneous, sublingual, oral, rectal, vaginal,parenteral, topical, pulmonary, intranasal, buccal, ophthalmic,intrathecal or another route of administration. Other contemplatedformulations include projected nanoparticles, liposomal preparations,resealed erythrocytes containing the active ingredient, andimmunologically based formulations.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in bulk, as a single unit dose, or as a plurality of single unitdoses. As used herein, a “unit dose” is a discrete amount of thepharmaceutical composition comprising a predetermined amount of theactive ingredient. The amount of the active ingredient is generallyequal to the dosage of the active ingredient that would be administeredto a subject or a convenient fraction of such a dosage such as, forexample, one-half or one-third of such a dosage.

The relative amounts of the active ingredient, the pharmaceuticallyacceptable carrier, and any additional ingredients in a pharmaceuticalcomposition of the invention will vary, depending upon the identity,size, and condition of the subject treated and further depending uponthe route by which the composition is to be administered. By way ofexample, the composition may comprise between 0.1% and 100% (w/w) activeingredient.

Controlled- or sustained-release formulations of a pharmaceuticalcomposition of the invention may be made using conventional technology.Formulations suitable for topical administration include, but are notlimited to, liquid or semi-liquid preparations such as liniments,lotions, oil-in-water or water-in-oil emulsions such as creams,ointments or pastes, and solutions or suspensions. Topicallyadministrable formulations may, for example, comprise from about 1% toabout 10% (w/w) active ingredient, although the concentration of theactive ingredient may be as high as the solubility limit of the activeingredient in the solvent. Formulations for topical administration mayfurther comprise one or more of the additional ingredients describedherein.

Enhancers of permeation may be used. These materials increase the rateof penetration of drugs across the skin. Typical enhancers in the artinclude ethanol, glycerol monolaurate, PGML (polyethylene glycolmonolaurate), dimethylsulfoxide, and the like. Other enhancers includeoleic acid, oleyl alcohol, ethoxydiglycol, laurocapram, alkanecarboxylicacids, polar lipids, or N-methyl-2-pyrrolidone.

One acceptable vehicle for topical delivery of some of the compositionsof the invention may contain liposomes. The composition of the liposomesand their use are known in the art (for example, see Constanza, U.S.Pat. No. 6,323,219).

The source of active compound to be formulated will generally dependupon the particular form of the compound. Small organic molecules andpeptidyl or oligo fragments can be chemically synthesized and providedin a pure form suitable for pharmaceutical usage. Products of naturalextracts can be purified according to techniques known in the art.Recombinant sources of compounds are also available to those of ordinaryskill in the art.

In alternative embodiments, the topically active pharmaceuticalcomposition may be optionally combined with other ingredients such asadjuvants, anti-oxidants, chelating agents, surfactants, foaming agents,wetting agents, emulsifying agents, viscosifiers, buffering agents,preservatives, and the like. In another embodiment, a permeation orpenetration enhancer is included in the composition and is effective inimproving the percutaneous penetration of the active ingredient into andthrough the stratum corneum with respect to a composition lacking thepermeation enhancer. Various permeation enhancers, including oleic acid,oleyl alcohol, ethoxydiglycol, laurocapram, alkanecarboxylic acids,dimethylsulfoxide, polar lipids, or N-methyl-2-pyrrolidone, are known tothose of skill in the art. In another aspect, the composition mayfurther comprise a hydrotropic agent, which functions to increasedisorder in the structure of the stratum corneum, and thus allowsincreased transport across the stratum corneum. Various hydrotropicagents such as isopropyl alcohol, propylene glycol, or sodium xylenesulfonate, are known to those of skill in the art.

The topically active pharmaceutical composition should be applied in anamount effective to affect desired changes. As used herein “amounteffective” shall mean an amount sufficient to cover the region of skinsurface where a change is desired. An active compound should be presentin the amount of from about 0.0001% to about 15% by weight volume of thecomposition. More preferable, it should be present in an amount fromabout 0.0005% to about 5% of the composition; most preferably, it shouldbe present in an amount of from about 0.001% to about 1% of thecomposition. Such compounds may be synthetically- or naturally derived.

Liquid derivatives and natural extracts made directly from biologicalsources may be employed in the compositions of this invention in aconcentration (w/v) from about 1 to about 99%. Fractions of naturalextracts and protease inhibitors may have a different preferred range,from about 0.01% to about 20% and, more preferably, from about 1% toabout 10% of the composition. Of course, mixtures of the active agentsof this invention may be combined and used together in the sameformulation, or in serial applications of different formulations.

The composition of the invention may comprise a preservative from about0.005% to 2.0% by total weight of the composition. The preservative isused to prevent spoilage in the case of an aqueous gel because ofrepeated patient use when it is exposed to contaminants in theenvironment from, for example, exposure to air or the patient's skin,including contact with the fingers used for applying a composition ofthe invention such as a therapeutic gel or cream. Examples ofpreservatives useful in accordance with the invention included but arenot limited to those selected from the group consisting of benzylalcohol, sorbic acid, parabens, imidurea and combinations thereof. Aparticularly preferred preservative is a combination of about 0.5% to2.0% benzyl alcohol and 0.05% to 0.5% sorbic acid.

The composition preferably includes an antioxidant and a chelating agentwhich inhibit the degradation of the compound for use in the inventionin the aqueous gel formulation. Preferred antioxidants for somecompounds are BHT, BHA, α-tocopherol and ascorbic acid in the preferredrange of about 0.01% to 0.3% and more preferably BHT in the range of0.03% to 0.1% by weight by total weight of the composition. Preferably,the chelating agent is present in an amount of from 0.01% to 0.5% byweight by total weight of the composition. Particularly preferredchelating agents include edetate salts (e.g., disodium edetate) andcitric acid in the weight range of about 0.01% to 0.20% and morepreferably in the range of 0.02% to 0.10% by weight by total weight ofthe composition. The chelating agent is useful for chelating metal ionsin the composition which may be detrimental to the shelf life of theformulation. While BHT and disodium edetate are the particularlypreferred antioxidant and chelating agent respectively for somecompounds, other suitable and equivalent antioxidants and chelatingagents may be substituted therefore as would be known to those skilledin the art. Controlled-release preparations may also be used and themethods for the use of such preparations are known to those of skill inthe art.

In some cases, the dosage forms to be used can be provided as slow orcontrolled-release of one or more active ingredients therein using, forexample, hydropropylmethyl cellulose, other polymer matrices, gels,permeable membranes, osmotic systems, multilayer coatings,microparticles, liposomes, or microspheres or a combination thereof toprovide the desired release profile in varying proportions. Suitablecontrolled-release formulations known to those of ordinary skill in theart, including those described herein, can be readily selected for usewith the pharmaceutical compositions of the invention. Thus, single unitdosage forms suitable for oral administration, such as tablets,capsules, gelcaps, and caplets that are adapted for controlled-releaseare encompassed by the present invention.

Most controlled-release pharmaceutical products have a common goal ofimproving drug therapy over that achieved by their non-controlledcounterparts. Ideally, the use of an optimally designedcontrolled-release preparation in medical treatment is characterized bya minimum of drug substance being employed to cure or control thecondition in a minimum amount of time. Advantages of controlled-releaseformulations include extended activity of the drug, reduced dosagefrequency, and increased patient compliance. In addition,controlled-release formulations can be used to affect the time of onsetof action or other characteristics, such as blood level of the drug, andthus can affect the occurrence of side effects.

Most controlled-release formulations are designed to initially releasean amount of drug that promptly produces the desired therapeutic effect,and gradually and continually release of other amounts of drug tomaintain this level of therapeutic effect over an extended period oftime. In order to maintain this constant level of drug in the body, thedrug must be released from the dosage form at a rate that will replacethe amount of drug being metabolized and excreted from the body.

Controlled-release of an active ingredient can be stimulated by variousinducers, for example pH, temperature, enzymes, water, or otherphysiological conditions or compounds. The term “controlled-releasecomponent” in the context of the present invention is defined herein asa compound or compounds, including, but not limited to, polymers,polymer matrices, gels, permeable membranes, liposomes, or microspheresor a combination thereof that facilitates the controlled-release of theactive ingredient.

Liquid suspensions may be prepared using conventional methods to achievesuspension of the active ingredient in an aqueous or oily vehicle.Aqueous vehicles include, for example, water, and isotonic saline. Oilyvehicles include, for example, almond oil, oily esters, ethyl alcohol,vegetable oils such as arachis, olive, sesame, or coconut oil,fractionated vegetable oils, and mineral oils such as liquid paraffin.Liquid suspensions may further comprise one or more additionalingredients including, but not limited to, suspending agents, dispersingor wetting agents, emulsifying agents, demulcents, preservatives,buffers, salts, flavorings, coloring agents, and sweetening agents. Oilysuspensions may further comprise a thickening agent. Known suspendingagents include, but are not limited to, sorbitol syrup, hydrogenatededible fats, sodium alginate, polyvinylpyrrolidone, gum tragacanth, gumacacia, and cellulose derivatives such as sodium carboxymethylcellulose,methylcellulose, hydroxypropylmethylcellulose. Known dispersing orwetting agents include, but are not limited to, naturally-occurringphosphatides such as lecithin, condensation products of an alkyleneoxide with a fatty acid, with a long chain aliphatic alcohol, with apartial ester derived from a fatty acid and a hexitol, or with a partialester derived from a fatty acid and a hexitol anhydride (e.g.,polyoxyethylene stearate, heptadecaethyleneoxycetanol, polyoxyethylenesorbitol monooleate, and polyoxyethylene sorbitan monooleate,respectively). Known emulsifying agents include, but are not limited to,lecithin, and acacia. Known preservatives include, but are not limitedto, methyl, ethyl, or n-propyl-para-hydroxybenzoates, ascorbic acid, andsorbic acid. Known sweetening agents include, for example, glycerol,propylene glycol, sorbitol, sucrose, and saccharin. Known thickeningagents for oily suspensions include, for example, beeswax, hardparaffin, and cetyl alcohol.

Liquid solutions of the active ingredient in aqueous or oily solventsmay be prepared in substantially the same manner as liquid suspensions,the primary difference being that the active ingredient is dissolved,rather than suspended in the solvent. Liquid solutions of thepharmaceutical composition of the invention may comprise each of thecomponents described with regard to liquid suspensions, it beingunderstood that suspending agents will not necessarily aid dissolutionof the active ingredient in the solvent. Aqueous solvents include, forexample, water, and isotonic saline. Oily solvents include, for example,almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis,olive, sesame, or coconut oil, fractionated vegetable oils, and mineraloils such as liquid paraffin.

Powdered and granular formulations of a pharmaceutical preparation ofthe invention may be prepared using known methods. Such formulations maybe administered directly to a subject, used, for example, to formtablets, to fill capsules, or to prepare an aqueous or oily suspensionor solution by addition of an aqueous or oily vehicle thereto. Each ofthese formulations may further comprise one or more of dispersing orwetting agent, a suspending agent, and a preservative. Additionalexcipients, such as fillers and sweetening, flavoring, or coloringagents, may also be included in these formulations.

A pharmaceutical composition of the invention may also be prepared,packaged, or sold in the form of oil-in-water emulsion or a water-in-oilemulsion. The oily phase may be a vegetable oil such as olive or arachisoil, a mineral oil such as liquid paraffin, or a combination of these.Such compositions may further comprise one or more emulsifying agentssuch as naturally occurring gums such as gum acacia or gum tragacanth,naturally-occurring phosphatides such as soybean or lecithinphosphatide, esters or partial esters derived from combinations of fattyacids and hexitol anhydrides such as sorbitan monooleate, andcondensation products of such partial esters with ethylene oxide such aspolyoxyethylene sorbitan monooleate. These emulsions may also containadditional ingredients including, for example, sweetening or flavoringagents.

As used herein, an “oily” liquid is one which comprises acarbon-containing liquid molecule and which exhibits a less polarcharacter than water.

A formulation of a pharmaceutical composition of the invention suitablefor oral administration may be prepared, packaged, or sold in the formof a discrete solid dose unit including, but not limited to, a tablet, ahard or soft capsule, a cachet, a troche, or a lozenge, each containinga predetermined amount of the active ingredient. Other formulationssuitable for oral administration include, but are not limited to, apowdered or granular formulation, an aqueous or oily suspension, anaqueous or oily solution, a paste, a gel, toothpaste, a mouthwash, acoating, an oral rinse, or an emulsion. The terms oral rinse andmouthwash are used interchangeably herein.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in a formulation suitable for oral or buccal administration.Such a formulation may comprise, but is not limited to, a gel, a liquid,a suspension, a paste, toothpaste, a mouthwash or oral rinse, and acoating. For example, an oral rinse of the invention may comprise acompound of the invention at about 1.4%, chlorhexidine gluconate(0.12%), ethanol (11.2%), sodium saccharin (0.15%), FD&C Blue No. 1(0.001%), peppermint oil (0.5%), glycerine (10.0%), Tween 60 (0.3%), andwater to 100%. In another embodiment, a toothpaste of the invention maycomprise a compound of the invention at about 5.5%, sorbitol, 70% inwater (25.0%), sodium saccharin (0.15%), sodium lauryl sulfate (1.75%),carbopol 934, 6% dispersion in (15%), oil of spearmint (1.0%), sodiumhydroxide, 50% in water (0.76%), dibasic calcium phosphate dihydrate(45%), and water to 100%. The examples of formulations described hereinare not exhaustive and it is understood that the invention includesadditional modifications of these and other formulations not describedherein, but which are known to those of skill in the art.

A tablet comprising the active ingredient may, for example, be made bycompressing or molding the active ingredient, optionally with one ormore additional ingredients. Compressed tablets may be prepared bycompressing, in a suitable device, the active ingredient in afree-flowing form such as a powder or granular preparation, optionallymixed with one or more of a binder, a lubricant, an excipient, a surfaceactive agent, and a dispersing agent. Molded tablets may be made bymolding, in a suitable device, a mixture of the active ingredient, apharmaceutically acceptable carrier, and at least sufficient liquid tomoisten the mixture. Pharmaceutically acceptable excipients used in themanufacture of tablets include, but are not limited to, inert diluents,granulating and disintegrating agents, binding agents, and lubricatingagents. Known dispersing agents include, but are not limited to, potatostarch and sodium starch glycollate. Known surface-active agentsinclude, but are not limited to, sodium lauryl sulphate. Known diluentsinclude, but are not limited to, calcium carbonate, sodium carbonate,lactose, microcrystalline cellulose, calcium phosphate, calcium hydrogenphosphate, and sodium phosphate. Known granulating and disintegratingagents include, but are not limited to, corn starch and alginic acid.Known binding agents include, but are not limited to, gelatin, acacia,pre-gelatinized maize starch, polyvinylpyrrolidone, and hydroxypropylmethylcellulose. Known lubricating agents include, but are not limitedto, magnesium stearate, stearic acid, silica, and talc.

Tablets may be non-coated or they may be coated using known methods toachieve delayed disintegration in the gastrointestinal tract of asubject, thereby providing sustained release and absorption of theactive ingredient. By way of example, a material such as glycerylmonostearate or glyceryl distearate may be used to coat tablets. Furtherby way of example, tablets may be coated using methods described in U.S.Pat. Nos. 4,256,108; 4,160,452; and 4,265,874 to form osmoticallycontrolled release tablets. Tablets may further comprise a sweeteningagent, a flavoring agent, a coloring agent, a preservative, or somecombination of these in order to provide for pharmaceutically elegantand palatable preparation.

Hard capsules comprising the active ingredient may be made using aphysiologically degradable composition, such as gelatin. Such hardcapsules comprise the active ingredient, and may further compriseadditional ingredients including, for example, an inert solid diluentsuch as calcium carbonate, calcium phosphate, or kaolin. Soft gelatincapsules comprising the active ingredient may be made using aphysiologically degradable composition, such as gelatin. Such softcapsules comprise the active ingredient, which may be mixed with wateror an oil medium such as peanut oil, liquid paraffin, or olive oil.

Liquid formulations of a pharmaceutical composition of the inventionwhich are suitable for oral administration may be prepared, packaged,and sold either in liquid form or in the form of a dry product intendedfor reconstitution with water or another suitable vehicle prior to use.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in a formulation suitable for rectal administration. Such acomposition may be in the form of, for example, a suppository, aretention enema preparation, and a solution for rectal or colonicirrigation.

Suppository formulations may be made by combining the active ingredientwith a non-irritating pharmaceutically acceptable excipient which issolid at ordinary room temperature (i.e., about 20° C.) and which isliquid at the rectal temperature of the subject (i.e., about 37° C. in ahealthy human). Suitable pharmaceutically acceptable excipients include,but are not limited to, cocoa butter, polyethylene glycols, and variousglycerides. Suppository formulations may further comprise variousadditional ingredients including, but not limited to, antioxidants, andpreservatives.

Retention enema preparations or solutions for rectal or colonicirrigation may be made by combining the active ingredient with apharmaceutically acceptable liquid carrier. As is well known in the art,enema preparations may be administered using, and may be packagedwithin, a delivery device adapted to the rectal anatomy of the subject.Enema preparations may further comprise various additional ingredientsincluding, but not limited to, antioxidants, and preservatives.

Methods for impregnating or coating a material with a chemicalcomposition are known in the art, and include, but are not limited tomethods of depositing or binding a chemical composition onto a surface,methods of incorporating a chemical composition into the structure of amaterial during the synthesis of the material (i.e., such as with aphysiologically degradable material), and methods of absorbing anaqueous or oily solution or suspension into an absorbent material, withor without subsequent drying.

As used herein, “parenteral administration” of a pharmaceuticalcomposition includes any route of administration characterized byphysical breaching of a tissue of a subject and administration of thepharmaceutical composition through the breach in the tissue. Parenteraladministration thus includes, but is not limited to, administration of apharmaceutical composition by injection of the composition, byapplication of the composition through a surgical incision, byapplication of the composition through a tissue-penetrating non-surgicalwound, and the like. In particular, parenteral administration iscontemplated to include, but is not limited to, intravenous,subcutaneous, intraperitoneal, intramuscular, intrasternal injection,and kidney dialytic infusion techniques.

Formulations of a pharmaceutical composition suitable for parenteraladministration comprise the active ingredient combined with apharmaceutically acceptable carrier, such as sterile water or sterileisotonic saline. Such formulations may be prepared, packaged, or sold ina form suitable for bolus administration or for continuousadministration. Injectable formulations may be prepared, packaged, orsold in unit dosage form, such as in ampules or in multi-dose containerscontaining a preservative. Formulations for parenteral administrationinclude, but are not limited to, suspensions, solutions, emulsions inoily or aqueous vehicles, pastes, and implantable sustained-release orbiodegradable formulations. Such formulations may further comprise oneor more additional ingredients including, but not limited to,suspending, stabilizing, or dispersing agents. In one embodiment of aformulation for parenteral administration, the active ingredient isprovided in dry (i.e., powder or granular) form for reconstitution witha suitable vehicle (e.g., sterile pyrogen-free water) prior toparenteral administration of the reconstituted composition.

The pharmaceutical compositions may be prepared, packaged, or sold inthe form of a sterile injectable aqueous or oily suspension or solution.This suspension or solution may be formulated according to the knownart, and may comprise, in addition to the active ingredient, additionalingredients such as antioxidants, dispersing agents, wetting agents, orsuspending agents described herein. Such sterile injectable formulationsmay be prepared using a non-toxic parenterally-acceptable diluent orsolvent, such as water or 1,3-butanediol, for example. Other acceptablediluents and solvents include, but are not limited to, Ringer'ssolution, isotonic sodium chloride solution, and fixed oils such assynthetic mono- or di-glycerides. Other parentally-administrableformulations which are useful include those which comprise the activeingredient in microcrystalline form, in a liposomal preparation, or as acomponent of a biodegradable polymer system. Compositions for sustainedrelease or implantation may comprise pharmaceutically acceptablepolymeric or hydrophobic materials such as an emulsion, an ion exchangeresin, a sparingly soluble polymer, or a sparingly soluble salt.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in a formulation suitable for buccal administration. Suchformulations may, for example, be in the form of tablets or lozengesmade using conventional methods, and may, for example, contain about 0.1to about 20% (w/w) active ingredient, the balance comprising an orallydissolvable or degradable composition and, optionally, one or more ofthe additional ingredients described herein. Alternately, formulationssuitable for buccal administration may comprise a powder or anaerosolized or atomized solution or suspension comprising the activeingredient. Such powdered, aerosolized, or aerosolized formulations,when dispersed, preferably have an average particle or droplet size inthe range from about 0.1 to about 200 nanometers, and may furthercomprise one or more of the additional ingredients described herein.

As used herein, “additional ingredients” include, but are not limitedto, one or more of the following: excipients; surface active agents;dispersing agents; inert diluents; granulating and disintegratingagents; binding agents; lubricating agents; sweetening agents; flavoringagents; coloring agents; preservatives; physiologically degradablecompositions such as gelatin; aqueous vehicles and solvents; oilyvehicles and solvents; suspending agents; dispersing or wetting agents;emulsifying agents, demulcents; buffers; salts; thickening agents;fillers; emulsifying agents; antioxidants; antibiotics; antifungalagents; stabilizing agents; and pharmaceutically acceptable polymeric orhydrophobic materials. Other “additional ingredients” which may beincluded in the pharmaceutical compositions of the invention are knownin the art and described, for example in Genaro, ed. (1985, Remington'sPharmaceutical Sciences, Mack Publishing Co., Easton, Pa.), which isincorporated herein by reference.

Typically, dosages of the compound of the invention which may beadministered to a subject, preferably a human, will vary depending uponany number of factors, including but not limited to, the type of animaland type of disease state being treated, the age of the subject and theroute of administration.

The compound can be administered to a subject as frequently as severaltimes daily, or it may be administered less frequently, such as once aday, once a week, once every two weeks, once a month, or even lessfrequently, such as once every several months or even once a year orless. The frequency of the dose will be readily apparent to the skilledartisan and will depend upon any number of factors, such as, but notlimited to, the type and severity of the disease being treated, the typeand age of the subject, etc.

Throughout this disclosure, various aspects of this invention can bepresented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual and partialnumbers within that range, for example, 1, 2, 3, 4, 5, 5.5 and 6. Thisapplies regardless of the breadth of the range.

It is contemplated that any embodiment discussed in this specificationcan be implemented with respect to any method or composition of theinvention, and vice versa. Furthermore, compositions of the inventioncan be used to achieve methods of the invention.

EXPERIMENTAL EXAMPLES

The invention is now described with reference to the following Examples.

These Examples are provided for the purpose of illustration only and theinvention should in no way be construed as being limited to theseExamples, but rather should be construed to encompass any and allvariations which become evident as a result of the teaching providedherein.

Without further description, it is believed that one of ordinary skillin the art can, using the preceding description and the followingillustrative examples, make and utilize the compounds of the presentinvention and practice the claimed methods. The following workingexamples therefore, specifically point out the preferred embodiments ofthe present invention, and are not to be construed as limiting in anyway the remainder of the disclosure.

The Materials and Methods are now described.

Exemplary compounds of the invention are set forth in Table 1. Table 1also identifies benzoxazoles used to prepare the exemplary compounds ofthe invention, as well as the starting material used to prepare thesebenzoxazoles. Commercially available compounds do not include compoundnumbers under the structure.

TABLE 1 Exemplary Compounds Compound Starting Material Benzoxazole ofThe Invention Name N/A

1 (S)-2-(2- hydroxyphenyl- amino)- thiazoline-5,5- dimethyl-4-carboxylic acid N/A

2 (R)-2-(2- hydroxyphenyl- amino)- thiazoline-5,5- dimethyl-4-carboxylic acid

3

4 (R)-2-(2-Hydroxy-4- methoxyphenyl amino)- thiazoline-4- carboxylicacid

5

6 (R)-2-(4- Fluoro-2- hydroxyphenyl- amino)- thiazoline-4- carboxylicacid

7

8 (R)-2-(3,5- Dichloro-2- hydroxy-4- methylphenyl- amino)- thiazoline-4-carboxylic acid

9

10 (R)-2-(5-tert- Butyl-2- hydroxyphenyl- amino)- thiazoline-4-carboxylic acid

11

12 (R)-2-(2- Hydroxy-4- methoxycarbon- ylphenylamino)- thiazoline-4-carboxylic acid

13

14 (R)-2-(5- Ethanesulfonyl- 2-hydroxy- phenylamino)- thiazoline-4-carboxylic acid

15

16 (R)-2-(4- Chloro-2- hydroxyphenyl- amino)- thiazoline-4- carboxylicacid

17

18 (R)-2-(2- Hydroxy-5- methoxyphenyl amino)- thiazoline-4- carboxylicacid

19

20 (S)-2-(2- Hydroxy-5- chlorophenyl- amino)-thiazoline- 5,5-dimethyl-4-carboxylic acid methyl ester

19

21 (S)-2-(2- Hydroxy-5- chlorophenyl- amino)- thiazoline-5,5-dimethyl-4- carboxylic acid

22

23 (S)-2-(2- Hydroxy-5- methylphenyl- amino)- thiazoline-5,5-dimethyl-4- carboxylic acid methyl ester

22

24 (S)-2-(2- Hydroxy-5- methylphenyl- amino)- thiazoline-5,5-dimethyl-4- carboxylic acid

25

26 (S)-2-(2- Hydroxy-4- methoxyphenyl amino)- thiazoline-5,5-dimethyl-4- carboxylic acid

25

27 (S)-2-(2- Hydroxy-4- methoxyphenyl amino)- thiazoline-5,5-dimethyl-4- carboxylic acid methyl ester

15

28 (S)-2-(4- Chloro-2- hydroxyphenyl- amino)- thiazoline-5,5-dimethyl-4- carboxylic acid

29

30 (S)-2-(2- Hydroxy-5- nitrophenyl- amino)- thiazoline- 5,5-dimethyl-4-carboxylic acid methyl ester

29

31 (S)-2-(2- Hydroxy-5- nitrophenyl- amino)- thiazoline- 5,5-dimethyl-4-carboxylic acid

13

32 (S)-2-(5- Ethanesulfonyl- 2-hydroxyphenyl- amino)- thiazoline-5,5-dimethyl-4- carboxylic acid methyl ester

13

33 (S)-2-(5- Ethanesulfonyl- 2-hydroxyphenyl- amino)- thiazoline-5,5-dimethyl-4- carboxylic acid N/A

34 (R)-2-(2- hydroxyphenyl- amino)- thiazoline-4- carboxylic acid methylester N/A

35 (S)-2-(2- hydroxyphenyl- amino)- thiazoline-5,5- dimethyl-4-carboxylic acid methyl ester

15

36 (R)-2-(4-chloro- 2-hydroxyphenyl- amino)- thiazoline-4- carboxylicacid methyl ester

15

37 (S)-2-(4-chloro- 2-hydroxyphenyl- amino)- thiazoline-5,5- dimethyl-4-carboxylic acid methyl ester

15

38 (R)-2-(4-chloro- 2-hydroxyphenyl- amino)- thiazoline-4- carboxylicacid N/A

39 (R)-2-(2- hydroxyphenyl- amino)- thiazoline-5,5- dimethyl-4-carboxylic acid methyl ester

Benzoxazole Formation Procedures: Compound 3:

A solution of 3-methoxyphenol (10 g, 80.55 mmol) in propionic acid (80mL) was treated at −5° C. with a solution of NaNO₂ (5.61 g, 81.30 mmol)in water (13 mL). After stirring for 1 h at −5° C., nitric acid (6.7 mL,161.10 mmol) was added. The slurry was stirred for 1 h at −5° C., andthen at room temperature for 16 h. Water (80 mL) was then added in adropwise fashion at room temperature. The resultant solid was filtered,washed with 50% aqueous propionic acid, and dried to give 7.47 g (55%)of 5-methoxy-2-nitro-phenol.

Subsequently, a mixture of 5-methoxy-2-nitrophenol (7.47 g) and 10% Pd/C(1.87 g) was prepared in an ethyl acetate/ethanol (100 mL, 1:1 v/v)mixture. The resulting slurry was stirred under an H₂ atmosphere for 3.5h at room temperature. Hydrogen was subsequently purged from thereaction flask and the reaction mixture was filtered through Celite.After washing the celite pad with additional solvent, volatiles in theresultant filtrate were removed in vacuo to give 2-amino-5-methoxyphenol(6.03 g, 98%).

Next, a mixture of 2-amino-5-methoxyphenol (3c) (6.03 g, 43.33 mmol) andpotassium ethyl xanthate (6.95 g, 73.33 mmol) in ethanol (80 mL) washeated at reflux for 18 h. The reaction was subsequently cooled and thesolvent was removed in vacuo. The residue was taken up in ethyl acetate(150 mL), washed successively with 4N HCl, water, and brine. The organicsolution was subsequently dried over Na₂SO₄. After filtration to removethe drying aid, the solvent was evaporated to give 6.23 g (79%) of6-methoxybenzoxazole-2-thiol.

Finally, a mixture of 6-methoxybenzoxazole-2-thiol (5.16 g, 28.48 mmol),thionyl chloride (28 mL) and catalytic amount of DMF (60 μL) was stirredat room temperature for 4 h. Volatiles were removed in vacuo. Toluene(30 mL) was added to the residue and the mixture was evaporated. Thisprocedure was repeated two more times to give crude compound 3, namely,2-chloro-6-methoxybenzoxazole.

Compound 5:

Compound 5 was prepared in accordance with the general procedures setforth for compound 3, except that the starting material was5-fluoro-2-nitrophenol and no nitration was performed.

Compound 7:

Compound 7 was prepared in accordance with the procedures set forth forcompound 3, except that the starting material was6-amino-2,4-chloro-3-methyl-phenol and no nitration or reduction wereperformed.

Compound 9:

Compound 9 was prepared in accordance with the procedure set forth forcompound 3, except that the starting material was2-amino-4-tert-butylphenol and no nitration or reduction were performed.

Compound 11:

Compound 11 was prepared in accordance with the procedure set forth forcompound 3, except that the starting material was4-amino-3-hydroxybenzoic acid methyl ester and no nitration or reductionwere performed.

Compound 13:

Compound 13 was prepared in accordance with the procedures set forth forcompound 3, except that the starting material was2-amino-4-ethanesulfonylphenol and no nitration or reduction wereperformed.

Compound 15:

Compound 15 was prepared in accordance with the procedures set forth forcompound 3, except that the starting material was 2-amino-5-chlorophenoland no nitration or reduction were performed.

Compound 17:

Compound 17 was prepared in accordance with the procedures set forth forcompound 3, except that the starting material was4-methoxy-2-nitrophenol and no nitration was performed.

Compound 19:

A mixture of potassium ethyl xanthate (13.4 g, 84.0 mmol) and4-chloro-2-aminophenol (10 g, 69.6 mmol) was dissolved in absoluteethanol (200 mL). The mixture was stirred under reflux overnight. Aftercooling, the solvent was evaporated under reduced pressure, the residuewas dissolved in water (200 mL) and treated with HOAc to adjust pH to 5.A solid formed, which was filtered and dried to afford5-chloro-2-mercaptobenzoxazole (12.8 g, yield 99.1%).

Subsequently, 5-chloro-2-mercaptobenzoxazole (3.1 g, 16.7 mmol) wasdissolved in thionyl chloride (30 mL, 413 mmol). DMF (1.5 mL) was addedand the reaction mixture was heated at 65° C. for 45 min. The solventwas removed under reduced pressure and to the residue was added toluene(2×60 mL) followed by evaporation each time to remove the excess SOCl₂(azetrope). The resultant crude product was dissolved in ethyl acetate(100 mL), washed with water (100 mL) and dried over Na₂SO₄. Evaporationof ethyl acetate gave 2,5-dichlorobenzoxazole, compound 19, as a red oil(3.2 g).

Compound 22:

A mixture of potassium ethyl xanthate (16.0 g, 0.1 mol) and2-amino-4-methylphenol (12.3 g, 0.1 mol) was dissolved in absoluteethanol (150 mL). The mixture was stirred at reflux overnight. Aftercooling, the reaction solvent was evaporated under reduced pressure. Theresultant residue was dissolved in water (200 mL) and treated with HOActo pH to 5. The resultant solid was filtered and dried to afford5-methyl-2-mercaptobenzoxazole (16.0 g, 96% yield).

Subsequently, the 5-methyl-2-mercaptobenzoxazole (6.2 g, 38 mmol) wasdissolved in thionyl chloride (36.9 mL) and 2.79 mL of DMF was added.The reaction mixture was then stirred at r.t. for 30 min. The solventwas removed under reduced pressure and to the residue was added toluene(2×60 mL) followed by evaporation each time to remove excess SOCl₂ viaan azetrope. The resultant crude product was dissolved in ethyl acetate(100 mL),washed with water (100 mL) and dried over Na₂SO₄. Evaporationof ethyl acetate gave crude 22 which was purified by silica-gel columnchromatography (eluent: petroleum ether) to provide pure2-chloro-5-methylbenzoxazole, compound 22, (3.5 g, 58%).

Compound 25:

To a solution of 3-methoxyphenol (11.1 g, 0.09 mmol) in propionic acid(90 mL) at 0° C., was added NaNO₂ (6.3 g, 0.09 mmol, in 5 mL water)dropwise. The mixture was stirred at 0° C. for 1 hour. Concentratednitric acid (12 mL) was then added dropwise and the mixture was stirredfor 1 hour at 0° C. and 2 hour at r.t. Water (30 mL) was subsequentlyadded and the resulting precipitate was collected and washed with 50%propionic acid, giving 2-nitro-5-methoxyphenol as yellow solid (5.8 g,40% yield).

Subsequently, a solution of the crude 2-nitro-5-methoxyphenol (5.0 g,29.6 mmol) in MeOH (150 mL) was prepared to which Pd/C (1.0 g) was thenadded. The mixture was placed under an H₂ atmosphere for about 2-3hours. After this time, the catalyst was removed via filtration andfiltrate was concentrated to give 2-amino-5-methoxyphenol (4.0 g, 97%yield).

Next, a mixture of potassium ethyl xanthate (4.6 g, 28.8 mmol) and crude2-amino-5-methoxyphenol (4.0 g, 28.8 mmol) was dissolved in ethanol (150mL). The mixture was heated at 80° C. overnight. After cooling, thesolvent was evaporated under reduced pressure. The resultant residue wasdissolved in water and treated with 1N HCl to pH 5, resulting in aprecipitate, which was collected. The solid thus collected was purifiedby chromatography with petroleum ether/ethyl acetate (3:1 v/v) to afford2-mercapto-6-methoxybenzoxazole (4.0 g, yield 77%).

Finally, pure 2-mercapto-6-methoxybenzoxazole (4.0 g, 22.2 mmol) wasdissolved in thionyl chloride (15 mL). DMF (1 mL) was added to thesolution and the reaction mixture was stirred at r.t for 10 min. Thesolvent was removed under reduced pressure. The crude product wasdissolved in ethyl acetate, washed with water, dried over Na₂SO₄, andconcentrated to give crude 2-chloro-6-methoxybenzoxazole, which waspurified by chromatography with petroleum ether/ethylacetate (3:1 v/v)to afford 2-chloro-6-methoxybenzoxazole, compound 25, (3.7 g, yield92%).

Compound 29:

A mixture of 2-amino-4-nitrophenol (12.5 g, 71 mmol) and potassium ethylxanthate (13 g, 81 mmol) were dissolved in ethanol (200 mL). Thereaction was heated at reflux overnight. After cooling, solvent in thereaction mixture was evaporated under reduced pressure. The resultantresidue was dissolved in water and adjusted to pH 2-3 with 2N HCl,resulting in the formation of a red solid. The red solid was filteredgiving 2-mercapto-5-nitrobenzoxazole (10.6 g, 76%).

Subsequently, the crude 2-mercapto-5-nitrobenzoxazole (10 g, 51 mmol),previously prepared, was dissolved in thionyl chloride (100 mL). DMF (60μL) was added and the reaction mixture was heated at 65° C. for 45 min.After cooling, the solvent was removed under reduced pressure and to theresidue, toluene was added twice (2×20 mL) and each time subsequentlyevaporated in vacuo to remove volatiles via azeotrope. The resultantcrude product was dissolved in ethyl acetate (400 mL), washed with brine(200 mL), dried over Na₂SO₄, filtered, and concentrated to give compound29, 2-chloro-5-nitrobenzoxazole, as (8.2 g, crude).

Preparation of Compounds According to the Invention: PurificationConditions:

Where specified, compounds of the invention were purified by reversephase chromatography eluting with a water-acetonitrite gradientcontaining either 0.05% trifluoroacetic acid or 0.05% aqueous ammonia(NH₄OH). Accordingly compounds were obtained as either atrifluoroacetate salt or as a free base.

Where specified, esters were purified using normal phase HPLC using anethyl acetate/petroleum ether gradient.

Where stated, compounds were isolated via dissolution in methanolfollowed by the addition of ethyl ether to induceprecipitation/crystallization.

Compound 1:

To a solution of D-(−)-penicillamine (6.2 g, 41.5 mmol) and DIPEA (14mL, 78.5 mmol) in 150 mL of MeOH/THF (1:2 v/v), was added2-chlorobenzoxazole (7.4 g, 48.0 mmol) in a dropwise manner. Thereaction was stirred overnight. The mixture was then concentrated invacuo to afford a pale yellow oil, which was treated with 150 mL ofconcentrated HCl to produce a white solid. This product was filtered,washed with acetone (2×50 mL) and Et₂O (3×50 mL) to yield(S)-2-(2-hydroxyphenylimino)-thiazolidine-5,5-dimethyl-4-carboxylic acidhydrochloride as a white solid (7.0 g, yield 56%).

Compound 2:

To a stirred solution of L-penicillamine (28.5 g, 0.19 mol) and2-chlorobenzoxazole (35.4 g, 0.23 mol) in 400 mL of MeOH/THF (3:1 (v/v))at r.t, was added N,N-diisopropylethylamine (“DIPEA”) (66.1 mL, 0.38mol) in a dropwise manner. The reaction was stirred overnight. Themixture was then concentrated at reduced pressure to afford pale yellowoil, which was formed white crystals when treated with concentrated HCl(300 mL). The white solid was filtered, washed with acetone (3×50 mL)and then with Et₂O (5×50 mL). The resultant white solid was crystallizedagain from MeOH/DCM, washed with DCM (3×50 mL) and Et₂O (2×50 mL) toyield compound 2 (as its HCl salt),(R)-2-(2-hydroxyphenylimino)-thiazolidine-5,5-dimethyl-4-carboxylic acidhydrochloride as a white solid (40.8 g, 71%). ¹H NMR (DMSO-d₆, ppm):δ=13.2 (br s (ex), 0.3H), 12.1 (br s (ex), 0.6H), 10.5 (br s, 1.4H),7.25 (m, 2H), 7.07 (d, 1H), 6.88 (t, 1H), 4.65 (br s, 1H), 1.72 (s, 3H),1.51 (s, 3H). ESI-MS: 267.1 (M⁺+H). HPLC purity: 95%.

Compound 4:

A solution of compound 3 (5.2 g, 28.32 mmol), (+)-S-trityl-L-cysteine(6) (9.34 g, 25.70 mmol) and DIPEA (6.4 mL, 38.68 mmol) in THF (25 mL)and methanol (40 mL) was stirred at room temperature for 3 h and then at40° C. for 14 h. Volatiles were subsequently removed in vacuo. Theresulting residue was suspended in water (60 mL) and acidified to pH 3with 5% KHSO₄ solution. The resulting suspension was extracted withCH₂Cl₂ (60 mL). The organic phase was washed with water, dried overNa₂SO₄, filtered, and the solvent removed in vacuo. The residue waspurified by flash column chromatography using gradient elution fromCH₂Cl₂/MeOH (99:1) to CH₂Cl₂/EtOH (9:1) to give(R)-2-(6-methoxybenzoxazol-2-ylamino)-3-tritylsulfanyl-propionic acid(6.40 g, 49%). 400 MHz 1H-NMR (DMSO-d₆, ppm): 13.1-12.7 (1H, br s) 8.23(1H, d, J=8.6 Hz) 7.38-7.19 (15H, m) 7.14 (1H, d, J=8.6 Hz) 7.08 (1H, d,J=2.1 Hz) 6.74 (1H, dd, J=8.6, 2.1 Hz) 4.13-4.06 (1H, m) 3.75 (3H, s)2.75 (1H, dd, 9.5 Hz) 2.55-2.50 (1H, m). ESI-MS (m/z): 511 [M+H]⁺

Subsequently, a solution of(R)-2-(6-methoxybenzoxazol-2-ylamino)-3-tritylsulfanyl-propionic acid(4.0 g, 7.83 mmol) in CH₂Cl₂ (35 mL) was prepared. Et₃SiH (4.2 mL, 25.8mmol) was added to the solution, followed by TFA (6.0 mL, 78.3 mmol).The reaction mixture was stirred under argon at 0° C. for 30 min. Thevolatiles were removed in vacuo and the residue was purified by flashcolumn chromatography using gradient elution from CH₂Cl₂/MeOH (99:1) toCH₂Cl₂/EtOH (1:2) to give compound compound 4 (1.2 g, 57%) as free aminoacid.

The resulting free amino acid (680 mg, 2.53 mmol) was dissolved in THF(5.0 ml) and 1.1 equivalents of 2M HCl in Et₂O (1.4 ml, 2.78 mmol) wasadded. The volatiles were removed in vacuo to give compound 4 as its HClsalt. 400 MHz 1H-NMR (DMSO-d6, ppm) 10.8-9.2 (2H, brs) 7.08 (1H, d,J=8.6 Hz) 6.39 (1H, d, J=2.7 Hz) 6.36 (1H, dd, J=8.6, 2.7 Hz) 4.82-4.76(1H, m) 3.67 (3H, s) 3.63 (1H, dd, 9.0 Hz) 3.51 (1H, dd, J=11.0, 5.7Hz). ESI-MS (m/z): 269 [M+H]⁺.

Compound 6:

A solution of compound 5 (4.24 g, 24.71 mmol), (+)—S-trityl-L-cysteine(8.16 g, 22.45 mmol) and DIPEA (5.6 mL, 33.67 mmol) in THF (20 mL) andmethanol (35 mL) was stirred at room temperature for 24 h. Volatileswere removed in vacuo. The residue was suspended in water and acidifiedto pH 3 with 5% KHSO₄ solution. The resulting suspension was extractedwith CH₂Cl₂ (2×100 mL). The organic phase was washed with water, driedover Na₂SO₄, filtered, and the solvent removed in vacuo. The residue waspurified by flash column chromatography using gradient elution fromCH₂Cl₂/MeOH (99:1) to CH₂Cl₂/EtOH (9:1) to give(R)-2-(6-fluorobenzoxazol-2-ylamino)-3-tritylsulfanyl-propionic acid(7.80 g, 70%). 400 MHz ¹H-NMR (DMSO-d₆, ppm): 8.46 (1H, d, J=8.6 Hz)7.40 (1H, dd, J=8.4, 2.4 Hz) 7.35-7.20 (16H, m) 6.99 (1H, ddd, J=10.2,8.4, 2.4 Hz) 4.14-4.06 (1H, m) 2.76 (1H, dd, J=12.5, 9.6 Hz) 2.53 (1H,dd, J=12.5, 4.4 Hz). ESI-MS (m/z): 499 [M+H]⁺

Compound 6, as its HCl salt, was prepared in 83% yield from(R)-2-(6-fluorobenzoxazol-2-ylamino)-3-tritylsulfanyl-propionic acidaccording to the general procedures described for the preparation ofcompound 4. 400 MHz ¹H-NMR (DMSO-d₆, ppm): 12.3-10.8 (3H, br s) 7.27(1H, dd, J=8.7, 6.4 Hz) 6.87 (1H, dd, J=10.4, 2.0 Hz) 6.72 (1H, ddd,J=8.7, 8.7, 2.5 Hz) 4.98-4.90 (1H, m) 3.95-3.83 (1H, m) 3.68 (1H, dd,J=8.2, 3.5 Hz). ESI-MS (m/z): 257 [M+H]⁺. Melting point: 109-111° C.

Compound 8:

(R)-2-(5,7-Dichloro-6-methylbenzoxazol-2-ylamino)-3-tritylsulfanyl-propionicacid was prepared from compound 7 according to the general proceduresspecified for compounds 4 and 6. 400 MHz ¹H-NMR (DMSO-d₆, ppm):13.5-12.5 (1H, br s) 8.85 (1H, d, J=8.2 Hz) 7.36 (1H, s) 7.34-7.20 (15H,m) 4.11-4.03 (1H, m) 2.76 (1H, dd, J=12.6, 9.9 Hz) 2.54 (1H, dd, J=12.6Hz). ESI-MS (m/z): 563, 565, 567 [M+H]⁺.

Compound 8, as its HCl salt, was prepared in 50% yield from(R)-2-(5,7-dichloro-6-methylbenzoxazol-2-ylamino)-3-tritylsulfanyl-propionicacid according to the general procedures described for compounds 4 and6. 400 MHz 1H-NMR (DMSO-d₆, ppm): 11.4-9.7 (2H, br s) 7.36 (1H, s) 4.96(1H, dd, J=8.5, 3.4 Hz) 3.93-3.83 (1H, m) 3.70 (1H, dd, J=11.4, 3.4 Hz)2.40 (3H, s). ESI-MS (m/z): 321, 323, 325 [M+H]⁺. Melting point:205-207° C.

Compound 10:

(R)-2-(5-tert-Butylbenzoxazol-2-ylamino)-3-tritylsulfanyl-propionic acidwas prepared from compound 9 in accordance with the procedures specifiedfor compounds 4 and 6. 400 MHz ¹H-NMR (DMSO-d₆, ppm): 13.2-12.6 (1H, brs) 8.33 (1H, d, J=8.6 Hz) 7.36-7.21 (17H, m) 7.04 (1H, dd, J=8.4, 2.0Hz) 4.15-4.08 (1H, m) 2.78 (1H, dd, J=9.8, 12.5 Hz) 2.54 (1H, dd,J=12.5, 4.6 Hz) 1.30 (9H, s). ESI-MS (m/z): 537 [M+H]⁺.

Compound 10, as its HCl salt, was prepared in 70% yield from(R)-2-(5-tert-butylbenzoxazol-2-ylamino)-3-tritylsulfanyl-propionic acidusing the general procedure described for compounds 4 and 6. 400 MHz¹H-NMR (DMSO-d₆, ppm): 12.0-11.6 (1H, br s) 10.5-9.9 (2H, br s) 7.27(1H, dd, J=8.6, 2.5 Hz) 7.22 (1H, d, J=2.5 Hz) 6.98 (1H, d, J=8.6 Hz)5.08-4.84 (1H, m) 3.99-3.85 (1H, m) 3.75-3.65 (1H, m) 1.24 (9H, s).ESI-MS (m/z): 295 [M+H]⁺.

Compound 12:

(R)-2-(1-Carboxy-2-tritylsulfanyl-ethylamino)-benzoxazole-6-carboxylicacid methyl ester was prepared from compound 11 in accordance with theprocedures set forth for compounds 4 and 6. 400 MHz ¹H-NMR (DMSO-d₆,ppm): 13.3-12.8 (1H, br s) 8.89 (1H, d, J=8.4 Hz) 7.90 (1H, d, J=1.4 Hz)7.83 (1H, dd, J=8.4, 1.4 Hz) 7.36-7.18 (16H, m) 4.19-4.11 (1H, m) 3.84(3H, s) 2.79 (1H, dd, J=12.6, 9.8 Hz) 2.56 (1H, dd, J=12.6, 9.8 Hz).ESI-MS (m/z): 539 M+H]⁺.

Compound 12, as its HCl salt, was prepared in 75% yield from(R)-2-(1-carboxy-2-tritylsulfanyl-ethylamino)-benzoxazole-6-carboxylicacid methyl ester according to the procedures set forth for compounds 4and 6. 400 MHz 1H-NMR (DMSO-d₆, ppm) 11.9-10.2 (3H, br s) 7.62-7.57 (1H,m) 7.49-7.42 (2H, m) 4.96 (1H, dd, J=8.8, 3.5 Hz) 3.92-3.83 (1H, m) 3.84(3H, s) 3.68 (1H, dd, J=11.5, 3.5 Hz). ESI-MS (m/z): 297 [M+H]⁺. Meltingpoint: 118-120° C.

Compound 14:

(R)-2-(6-Ethanesulfonylbenzoxazol-2-ylamino)-3-tritylsulfanyl-propionicacid was prepared from compound 13 according to the general proceduresspecified for the preparation of compounds 4 and 6. 400 MHz ¹H-NMR(DMSO-d₆, ppm): 8.69-8.59 (1H, m) 7.67 (1H, d, J=1.8 Hz) 7.63 (1H, d,J=8.2 Hz) 7.55 (1H, dd, J=8.2, 1.8 Hz) 7.37-7.18 (15H, m) 4.13-4.05 (1H,m) 3.28 (2H, q, J=7.4 Hz, overlapped with water) 2.76 (1H, dd, J=12.2,9.2 Hz) 2.59 (1H, dd, J=12.2, 4.2 Hz) 1.09 (3H, t, J=7.4 Hz). ESI-MS(m/z): 573 [M+H]⁺.

Compound 14, as its HCl salt, was prepared in 33% yield from(R)-2-(6-ethanesulfonyl-benzoxazol-2-ylamino)-3-tritylsulfanyl-propionicacid as set forth for compounds 4 and 6. 400 MHz 1H-NMR (DMSO-d₆, ppm):12.4-11.5 (2H, br s) 7.81-7.71 (1H, m) 7.69-7.59 (1H, m) 7.15 (1H, d,J=8.4 Hz) 4.94-4.87 (1H, m) 3.87-3.76 (1H, m) 3.68-3.60 (1H, m) 3.17(2H, q, J=7.2 Hz) 1.06 (3H, t, J=7.2 Hz). ESI-MS (m/z): 331 [M+H]⁺.

Compound 16:

(R)-2-(6-Chlorobenzoxazol-2-ylamino)-3-tritylsulfanyl-propionic acid wasprepared from compound 15 in accordance with the general proceduresspecified for compounds 4 and 6. 400 MHz ¹H-NMR (DMSO-d₆, ppm): 8.50(1H, d, J=8.4 Hz) 7.39 (1H, d, J=8.4 Hz) 7.33-7.19 (16H, m) 7.03 (1H,dd, J=8.4, 2.1 Hz) 4.13-4.06 (1H, m) 2.74 (1H, dd, J=12.4, 9.3 Hz) 2.55(1H, dd, J=12.4, 4.6 Hz). ESI-MS (m/z): 515, 517 [M+H]⁺.

Compound 16 was prepared in 42% yield from(R)-2-(6-chlorobenzoxazol-2-ylamino)-3-tritylsulfanyl-propionic acidaccording to the general procedures described for compounds 4 and 6. 400MHz ¹H-NMR (DMSO-d₆, ppm): 12.0-10.4 (3H, br s) 7.46-7.37 (1H, m)7.26-7.17 (1H, m) 7.02 (1H, d, J=8.6 Hz) 4.94 (1H, dd, J=8.4, 3.5 Hz)3.91-3.80 (1H, m) 3.66 (1H, dd, J=11.5, 3.5 Hz). ESI-MS (m/z): 273, 275[M+H]⁺.

Compound 18:

(R)-2-(5-Methoxybenzoxazol-2-ylamino)-3-tritylsulfanyl-propionic acidwas prepared from compound 17 in accordance with the general proceduresspecified for compounds 4 and 6. 200 MHz ¹H-NMR (DMSO-d₆, ppm): 8.36(1H, d, J=8.6 Hz) 7.38-7.18 (16H, m) 6.86 (1H, d, J=2.6 Hz) 6.57 (1H,dd, J=8.8, 2.6 Hz) 4.17-4.02 (1H, m) 3.73 (3H, s) 2.75 (1H, dd, J=12.2,9.9 Hz) 2.57-2.50 (1H, m, overlapped with DMSO). ESI-MS (m/z): 511[M+H]⁺.

Compound 18, as its HCl salt, was prepared in 48% yield from(R)-2-(5-methoxybenzoxazol-2-ylamino)-3-tritylsulfanyl-propionic acidusing the general procedures described for the preparation of compounds4 and 6. 400 MHz 1H-NMR (DMSO-d₆, ppm): 12.2-9.5 (3H, m) 6.92 (1H, d,J=8.8 Hz) 6.90-6.86 (1H, m) 6.83-6.76 (1H, m) 4.96-4.89 (1H, m)3.90-3.83 (1H, m) 3.67 (3H, s) 3.68-3.63 (1H, m). ESI-MS (m/z): 269[M+H]⁺.

Compound 20:

Compound 19 (1.5 g, 8.3 mmol) and (S)-methyl2-amino-3-mercapto-3-methylbutanoate (1.7 g, 8.5 mmol) were dissolved in15 mL of DMF. Subsequently, DIPEA (2.9 mL) was added. The reactionmixture was stirred at r.t. for 3 h. The reaction was diluted with ethylacetate (200 mL) washed with water (100 mL), saturated NH₄Cl solution(100 mL) and brine solution (50 mL). The organic phase was dried overNa₂SO₄, filtered and concentrated to give a black oil, which was thenpurified by silica-gel column chromatography (eluent: i-PrOH /PE=1:20)to give a pale yellow solid. The solid was recrystallized from ethylacetate and heptane, giving compound 20 (725 mg, yield 27%, purity:99%). ¹H NMR (CDCl₃, 500 MHz): δ 6.96 (d, J=8.4 Hz, 1H), 6.95 (s, 1H),6.96 (d, J=7.5 Hz, 1H), 4.43 (s, 1H), 3.82 (s, 3H), 1.73 (s, 3H), 1.47(s, 3H). ¹³C NMR (CDCl₃, 125 MHz): δ 167.3, 160.3, 146.1, 123.1, 122.1,118.6, 50.6, 26.3, 23.7. ESI-MS (m/z): 315.0 [M+1]⁺.

Compound 21:

Compound 19 (1.65 g, 8.8 mmol) and D-penicillamine (1.40 g, 9.4 mmol)were dissolved in 10 mL of DMF. Subsequently, DIPEA (2.5 mL) was added.The resulting mixture was stirred at r.t. for 2 hours. The reaction wasthen diluted with water (200 mL) and washed with ethyl acetate (2×150mL). The aqueous phase was concentrated and the residue was purified byprep-HPLC eluting with an H₂O/CH₃CN gradient containing 0.05% TFA togive(S)-2-(2-hydroxy-5-chlorophenylamino)-thiazoline-5,5-dimethyl-4-carboxylicacid as a white solid TFA salt (1.2 g, yield 45%, purity: 100%). ¹H NMR(DMSO-d6, 400 MHz): δ 7.37 (s, 1H), 7.21 (d, J=8.0 Hz, 1H), 6.97 (d,J=8.4 Hz, 1H), 4.61 (brs, 1H), 1.70 (s, 3H), 1.49 (s, 3H); ¹³C NMR(DMSO-d6, 100 MHz): δ 168.6, 150.5, 128.3, 125.3, 122.1, 115.5, 113.3,72.2, 57.4, 28.5, 24.7; ESI-MS (m/z): 301.0 [M+1]⁺

Compound 23:

Compound 22 (668 mg, 4.0 mmol) and (S)-methyl2-amino-3-mercapto-3-methylbutanoate hydrochloride (960 mg, 4.8 mmol)were dissolved in 10 mL of DMF. Subsequently, DIPEA (8.0 mL) was added.The mixture was stirred at r.t. overnight. The reaction was diluted withethyl acetate (200 mL) and washed with water (100 mL). The organic phasewas dried over Na₂SO₄, filtered, and concentrated in vacuo. The residuewas purified by silica-gel column chromatography (eluent: ethylacetate/petroleum ether: 1:3-1:1) to obtain compound 23.

The crude product was further purified by prep-HPLC eluting with anH₂O/CH₃CN gradient containing 0.05% TFA to get(S)-2-(2-hydroxy-5-methylphenylamino)-thiazoline-5,5-dimethyl-4-carboxylicacid methyl ester TFA salt (600 mg, yield 51%). ¹H NMR (CDCl₃, 500 MHz):δ 6.92 (d, J=7.5 Hz, 1H), 6.71 (s, 1H), 6.56 (d, J=7.0 Hz, 1H), 4.58 (s,1H), 3.84 (s, 3H), 2.20 (s, 3H), 1.73 (s, 3H), 1.50 (s, 3H); ¹³C NMR(CDCl₃, 125 MHz): δ 174.3, 166.6, 151.4, 140.8, 125.3, 121.7, 120.6,118.1, 71.4, 57.5, 53.1, 28.5, 25.2, 21.2; ESI-MS (m/z): 295.1 [M+1]⁺.

Compound 24:

Compound 22 (1.0 g, 6.0 mmol) and (S)-penicillamine (983 mg, 6.6 mmol)were dissolved in 5 mL of DMF. Subsequently, DIPEA (1.55 mL, 12 mmol)was added. The reaction was stirred at r.t. overnight. The mixture wasconcentrated and the resultant residue was purified by prep-HPLC elutingwith an H₂O/CH₃CN gradient containing 0.05% TFA. The resultant productwas recrystallized from MeOH and diethyl ether to give compound 24 as asalt. ¹H NMR (DMSO-d6, 500 MHz): δ 6.99 (d, J=6.0 Hz, 1H), 6.61 (s, 1H),6.54 (d, J=7.5 Hz, 1H), 4.39 (s, 1H), 2.18 (s, 3H), 1.63 (s, 3H), 1.40(s, 3H); ¹³C NMR (DMSO-d6, 125 MHz): δ 170.8, 160.5, 148.5, 134.2,127.5, 121.9, 119.6, 118.1, 77.0, 58.7, 28.1, 25.5, 20.4; ESI-MS (m/z):281.1 [M+1]⁺.

Compound 26:

Compound 25 (870 mg, 4.7 mmol) and D-penicillamine (710 mg, 4.7 mmol)were dissolved in DMSO (20 mL), and NaOH (430 mg, 10.7 mmol) was added.The reaction was stirred at r.t. for 3 h. The mixture was then acidifiedwith 6N HCl (1.5 mL) to pH 3-4, then purified by flash chromatographyeluting with acetonitrile/distilled water (with 0.05% TFA) givingcompound 26 as a salt (1.0 g, 74%, purity: 97%). ¹H NMR (DMSO-d6, 500MHz): δ 6.97 (d, J=8.0 Hz, 1H), 6.37 (s, 1H), 6.34 (d, J=8.5 Hz, 1H),4.38 (s, 1H), 3.66 (s, 3H), 1.63 (s, 3H), 1.40 (s, 3H); ¹³C NMR(DMSO-d6, 125 MHz): δ 170.5, 157.8, 151.0, 124.1, 122.3, 104.5, 103.0,76.2, 58.7, 55.1, 27.7, 25.5; ESI-MS (m/z): 297.1 [M+1]⁺.

Compound 27:

A solution of compound 26 (800 mg, 2.7 mmol) in MeOH (30 mL) wasprepared. SOCl₂ (0.8 mL) was then added in a dropwise manner. Themixture was stirred overnight at r.t. The resulting solution wasconcentrated under reduced pressure to obtain the title compound aswhite solid compound 27 as its HCl salt (930 mg, 99%, purity: 95%). ¹HNMR (DMSO-d6, 500 MHz): δ 11.7 (brs, 1H), 10.5 (brs, 1H), 7.12 (d, J=8.0Hz, 1H), 6.37 (brs, 1H), 6.46 (brs, 1H), 4.74 (brs, 1H), 3.77 (s, 3H),3.72 (s, 3H), 1.70 (s, 3H), 1.43 (s, 3H); ¹³C NMR (DMSO-d6, 125 MHz): δ167.6, 160.2, 153.3, 127.5, 104.8, 102.2, 70.7, 57.0, 55.2, 52.6, 24.2;ESI-MS (m/z): 311.1 [M+1]⁺.

Compound 28:

Compound 15 (693 mg, 3.7 mmol) and D-penicillamine (550 mg, 3.7 mmol)were dissolved in 4 mL of DMF. Subsequently DIPEA (960 mg, 7.5 mmol) wasadded. The reaction was stirred at r.t. for 3 h. The resultant mixturewas concentrated and the crude product was purified by preparative HPLCeluting with an H₂O/CH₃CN gradient containing 0.05% TFA, giving compound28 as a pink solid (652 mg, yield 58%). ¹H NMR (DMSO-d6, 500 MHz): δ7.17 (brs, 1H), 6.81-6.75 (m, 2H), 4.37 (s, 1H), 1.62 (s, 3H), 1.39 (s,3H); ¹³C NMR (DMSO-d6, 125 MHz): δ 170.7, 159.4, 149.4, 127.2, 122.4,118.7, 116.5, 28.3, 25.5; ESI-MS (m/z): 301.1 [M+1]⁺

Compound 30:

To a solution of compound 29 (1.0 g, 5.0 mmol) in DMF (30 mL) was addedD-penicillamine methyl ester (1.6 g, 10.0 mmol) and Na₂CO₃ (2.5 g, 19.8mmol). The reaction was stirred at r.t. for 30 min. The resultantmixture was diluted with ethyl acetate (150 mL) and the resultingorganic phase was washed with NH₄Cl solution, brine, dried over Na₂SO₄,filtered and concentrated in vacuo. The resultant crude product waspurified by preparative HPLC eluting with an H₂O/CH₃CN gradientcontaining 0.05% TFA to obtain(R)-2-(2-hydroxy-5-nitrophenylamino)-thiazoline-5,5-dimethyl-4-carboxylicacid methyl ester (850 mg, 50%). ¹H NMR (CDCl₃, 500 MHz): δ 8.06 (s,1H), 7.98 (d, J=9.0 Hz, 1H), 7.01 (d, J=9.0 Hz, 1H), 4.64 (s, 1H), 3.93(s, 3H), 1.81 (s, 3H), 1.50 (s, 3H); ¹³C NMR (MeOD, 125 MHz): δ 173.6,168.7, 159.5, 141.6, 126.9, 125.2, 123.8, 117.7, 73.0, 59.6, 53.5, 29.3,25.1; ESI-MS (m/z): 326.1 [M+1]⁺.

Compound 31:

Compound 29 (1.84 g, 9.3 mmol) and D-penicillamine (1.4 g, 9.3 mmol)were dissolved in DMF (5 mL). Subsequently DIPEA (2 mL) was added. Thereaction mixture was stirred at r.t. overnight. The reaction was dilutedwith ethyl acetate (200 mL) and the solid was filtered off. Theresultant organic phase was washed with water and the resulting aqueousphases were combined and concentrated. The resulting residue waspurified by preparative HPLC eluting with an H₂O/CH₃CN gradientcontaining 0.05% TFA. Compound 31 was obtained as white solid (1.28 g,yield 45%, purity: 100%). ¹H NMR (DMSO-d6, 500 MHz): δ 8.24 (brs, 1H),8.05 (d, J=9.0 Hz, 1H), 7.09 (d, J=9.0 Hz, 1H), 4.59 (s, 1H), 1.70 (s,3H), 1.49 (s, 3H); ¹³C NMR (MeOD, 125 MHz): δ 169.3, 158.3, 139.8,124.5, 121.7, 118.4, 116.9, 115.5, 72.7, 57.8, 28.9, 25.3; ESI-MS (m/z):312.0 [M+1]⁺.

Compound 32:

Compound 13 (800 mg, 3.2 mmol) and D-penicillamine methyl ester (1.0 g,6.1 mmol) were dissolved in DMF (20 mL). Na₂CO₃ (500 mg, 4.7 mmol) wassubsequently added. The reaction was stirred at 0° C. for 2 h. Themixture was then diluted with ethyl acetate (100 mL) and subsequentlywashed with aqueous NH₄Cl (100 mL) and then with brine (50 mL). Theorganic phase was dried (NaSO₄) and concentrated. The resultant crudeproduct was purified by silica gel chromatography (eluent: ethylacetate/petroleum ether: 1:1 (v/v)) and the final product wasrecrystallized from ethyl acetate/petroleum ether giving compound 32 asa white solid (550 mg, 40%, 99% purity). ¹H NMR (CDCl₃, 500 MHz): δ7.53-7.52 (m, 2H), 7.98 (d, J=8.5 Hz, 1H), 4.44 (s, 1H), 3.84 (s, 3H),3.08 (q, J=7.5 Hz, 2H), 1.75 (s, 3H), 1.48 (s, 3H), 1.26 (t, J=7.5 Hz,3H); ¹³C NMR (MeOD, 125 MHz): δ 171.6, 164.0, 155.4, 129.6, 126.0,122.7, 117.3, 52.7, 51.6, 49.0, 29.3, 25.9, 7.8; ESI-MS (m/z): 373.0[M+1]⁺.

Compound 33:

Compound 13 (2.0 g, 8.1 mmol) and D-penicillamine (1.22 g, 8.1 mmol)were dissolved in DMF (20 mL) and DIPEA (3.3 g, 32.6 mmol) was added.The reaction was stirred at 0° C. for 15 min. The mixture was thenconcentrated and the resultant crude product was purified by preparativeHPLC eluting with an H₂O/CH₃CN gradient containing 0.05% TFA, givingcompound 33 as a white solid (380 mg, 30%). ¹H NMR (D₂O, 500 MHz): δ7.82 (s, 1H), 7.76 (d, J=9.0 Hz, 1H), 7.19 (d, J=9.0 Hz, 1H), 3.26 (q,J=7.5 Hz, 2H), 1.70 (s, 3H), 1.51 (s, 3H), 1.49 (t, J=7.5 Hz, 3H); ¹³CNMR (MeOD, 125 MHz): δ 158.4, 134.4, 128.3, 118.3, 73.6, 59.5, 51.4,29.0, 26.3, 7.7; ESI-MS (m/z): 359.0 [M+1]⁺.

Compound 34:

Compound 34 was prepared from 2-chlorobenzoxazole and L-cysteine methylester following the procedure used to prepare compound 2.

Compound 35:

Compound 35 was prepared from 2-chlorobenzoxazole and D-penicillaminemethyl ester according to the procedure described for compound 20.

Compound 36:

Compound 36 was prepared from 2,6-dichlorobenzoxazole (compound 15) andL-cysteine methyl ester following the procedure described for compound20.

Compound 37:

Compound 37 was prepared from 2,6-dichlorobenzoxazole (compound 15) andD-penicillamine methyl ester according to the procedure described forcompound 20.

Compound 38:

Compound 38 was prepared from 2,6-dichlorobenzoxazole (compound 15) andL-cysteine according to the procedure set forth for compound 21.

Compound 39:

Compound 39 was prepared from compound 2 according to the procedure setforth for compound 27.

Crystal Structure Data For Compound 6 Fractional Atomic Coordinates &U(ISO)

Atom x/a y/b z/c U(iso) S(1) 0.0619(5) 0.89329(18) 0.62592(5) 0.0443(10)F(14) −0.0622(11) 0.4166(4) 0.46571(10) 0.070(3) O(16) −0.3230(12)1.0220(5) 0.79075(13) 0.055(3) O(17) −0.4006(12) 1.1860(5) 0.73194(14)0.056(3) O(13) 0.1024(13) 0.3833(5) 0.63281(14) 0.068(3) N(6)−0.2559(14) 0.6449(5) 0.64916(13) 0.037(3) C(5) 0.0061(17) 1.0476(7)0.66746(17) 0.043(4) N(3) −0.2344(14) 0.8374(5) 0.70441(14) 0.037(3)C(2) −0.1638(15) 0.7796(7) 0.66208(17) 0.033(3) C(15) −0.2837(18)1.0732(7) 0.7486(2) 0.041(4) C(4) −0.0713(16) 0.9757(6) 0.71660(18)0.035(4) C(12) −0.3366(17) 0.6534(7) 0.56100(19) 0.050(4) C(10)−0.113(2) 0.4730(8) 0.5110(2) 0.054(5) C(7) −0.2030(17) 0.5848(7)0.60091(18) 0.040(4) C(11) −0.2924(18) 0.5958(8) 0.51475(19) 0.048(5)C(8) −0.0280(18) 0.4573(6) 0.5958(2) 0.043(4) C(9) 0.0169(17) 0.3990(7)0.54902(19) 0.051(5) H(5A) 0.22988 1.100461 0.665826 0.056664 H(5B)−0.16939 1.129391 0.655266 0.056664 H(6) −0.42047 0.595765 0.669080.061308 H(9) 0.136361 0.311578 0.542664 0.084648 H(11) −0.3840110.641693 0.486906 0.068208 H(12) −0.459431 0.740398 0.566439 0.078816H(13) 0.153817 0.41732 0.666581 0.143532 H(3) −0.337489 0.7737290.731041 0.059208 H(4) 0.131722 0.957573 0.738132 0.04524Temperature factor of the form: exp[−2π^(2U)], U=U(iso) or⅓ SUM(i)SUM(j){U(ij)*astar(i).astar(j).a(i).a(j). cos(ij)}

Anisotropic Thermal Parameters

Atom U11 U22 U33 U12 U13 U23 S(1) 0.0558(13) 0.0428(10) 0.0342(8)−.0013(10) 0.0127(8) −.0013(8) F(14) 0.104(4) 0.072(3) 0.0354(18)−.006(3) 0.011(2) −.0258(18) O(16) 0.079(4) 0.059(3) 0.026(2) 0.017(3)0.012(2) 0.005(2) O(17) 0.078(4) 0.049(3) 0.041(2) 0.025(3) 0.014(3)0.004(2) O(13) 0.120(5) 0.044(3) 0.041(2) 0.027(3) −.016(3) −.001(2)N(6) 0.051(4) 0.034(3) 0.027(2) −.004(3) 0.009(2) −.002(2) C(5) 0.047(5)0.044(4) 0.038(3) 0.008(4) 0.017(4) −.001(3) N(3) 0.049(4) 0.036(3)0.027(3) 0.009(3) 0.006(3) −.001(2) C(2) 0.037(5) 0.040(4) 0.023(3)−.003(3) 0.006(3) 0.003(3) C(15) 0.041(5) 0.041(4) 0.040(4) −.011(4)0.004(3) −.013(3) C(4) 0.038(5) 0.037(4) 0.031(3) −.001(4) 0.004(3)−.003(3) C(12) 0.066(6) 0.050(4) 0.033(3) 0.001(4) 0.005(3) −.001(3)C(10) 0.084(7) 0.055(4) 0.022(3) −.010(5) 0.003(4) −.009(3) C(7)0.055(5) 0.034(4) 0.030(3) −.013(4) −.001(3) −.001(3) C(11) 0.057(5)0.048(4) 0.038(3) −.003(5) 0.001(3) −.003(3) C(8) 0.057(6) 0.030(4)0.041(3) −.005(4) 0.002(4) −.004(3) C(9) 0.071(6) 0.037(4) 0.044(4)−.002(5) 0.004(4) −.020(3) T = exp[2π**2(U11.h**2.astar**2 +U22.k**2.bstar**2 + U33.l**2.cstar**2 + 2U12.h.k.astar.bstar +2U13.h.l.astar.cstar + 2U23.k.l.bstar.cstar)]

Intramolecular Bond Lengths

Minimum bond length=0.80 A: Maximum bond length=1.80 A

S(1)—C(2) 1.735(6) F(14)—C(10) 1.369(7) O(16)—C(15) 1.264(7) O(17)—C(15)1.226(8) O(13)—C(8) 1.346(7) N(6)—C(2) 1.327(8) N(6)—C(7) 1.458(6)C(5)—C(4) 1.542(7) N(3)—C(2) 1.317(7) N(3)—C(4) 1.473(8) C(15)—C(4)1.551(9) C(12)—C(7) 1.391(8) C(12)—C(11) 1.393(8) C(10)—C(11) 1.356(11)C(10)—C(9) 1.367(9) C(7)—C(8) 1.384(9) C(8)—C(9) 1.410(8) O(16)—H(6)1.70(4) O(16)—H(13) 1.68(4) O(17)—H(3) 1.72(5) O(13)—H(13) 1.00(4)N(6)—H(6) 1.00(5) C(5)—H(5A) 1.08(7) C(5)—H(5B) 1.11(7) N(3)—H(3)1.03(5) C(4)—H(4) 1.07(6) C(12)—H(12) 0.96(7) C(11)—H(11) 0.95(6)C(9)—H(9) 0.97(7)

Intramolecular Bond Angles (H Omitted)

Minimum bond length=0.80 A: Maximum bond length=1.80 A

C(2)—N(6)—C(7) 122.6(5) C(2)—N(3)—C(4) 115.3(5) S(1)—C(2)—N(6) 123.8(4)S(1)—C(2)—N(3) 114.1(5) N(6)—C(2)—N(3) 122.1(5) O(16)—C(15)—O(17)126.5(6) O(16)—C(15)—C(4) 113.5(5) O(17)—C(15)—C(4) 120.0(5)C(5)—C(4)—N(3) 105.0(4) C(5)—C(4)—C(15) 113.2(5) N(3)—C(4)—C(15)109.0(5) C(7)—C(12)—C(11) 120.3(6) F(14)—C(10)—C(11) 117.8(5)F(14)—C(10)—C(9) 117.1(6) C(11)—C(10)—C(9) 125.2(6) N(6)—C(7)—C(12)119.6(6) N(6)—C(7)—C(8) 119.2(5) C(12)—C(7)—C(8) 121.1(5)C(12)—C(11)—C(10) 117.0(6) O(13)—C(8)—C(7) 124.4(5) O(13)—C(8)—C(9)117.0(5) C(7)—C(8)—C(9) 118.6(5) C(10)—C(9)—C(8) 117.8(6)

Intramolecular Torsion Angles (H Omitted)

Minimum bond length=0.80 A: Maximum bond length=1.80 A

Bonds Angles C(7)—N(6)—C(2)—S(1) −6.6(4) C(2)—N(6)—C(7)—C(12) −62.8(7)C(4)—N(3)—C(2)—S(1) −9.3(4) C(2)—N(3)—C(4)—C(5) 27.2(5)S(1)—C(2)—N(6)—C(7) −6.6(4) N(6)—C(2)—N(3)—C(4) 169.8(8)O(16)—C(15)—C(4)—C(5) −173.3(8) O(17)—C(15)—C(4)—C(5) 8.3(6)C(5)—C(4)—N(3)—C(2) 27.2(5) C(5)—C(4)—C(15)—O(17) 8.3(6)N(3)—C(4)—C(15)—O(17) −108.1(7) C(11)—C(12)—C(7)—N(6) −178.9(9)C(11)—C(12)—C(7)—C(8) −0.9(6) F(14)—C(10)—C(9)—C(8) 178.7(9)C(11)—C(10)—C(9)—C(8) −2.5(7) N(6)—C(7)—C(12)—C(11) −178.9(9)C(8)—C(7)—N(6)—C(2) 119.2(8) C(12)—C(7)—C(8)—O(13) −178.5(10)C(12)—C(7)—C(8)—C(9) 1.0(6) C(10)—C(11)—C(12)—C(7) −0.7(6)O(13)—C(8)—C(7)—N(6) −0.5(5) O(13)—C(8)—C(9)—C(10) −179.8(9)C(9)—C(8)—C(7)—C(12) 1.0(6) C(8)—C(9)—C(10)—F(14) 178.7(9)C(10)—C(9)—C(8)—C(7) 0.7(6) C(7)—N(6)—C(2)—N(3) 174.4(9)C(2)—N(6)—C(7)—C(8) 119.2(8) C(4)—N(3)—C(2)—N(6) 169.8(8)C(2)—N(3)—C(4)—C(15) 148.7(7) S(1)—C(2)—N(3)—C(4) −9.3(4)N(3)—C(2)—N(6)—C(7) 174.4(9) O(16)—C(15)—C(4)—N(3) 70.2(6)O(17)—C(15)—C(4)—N(3) −108.1(7) C(5)—C(4)—C(15)—O(16) −173.3(8)N(3)—C(4)—C(15)—O(16) 70.2(6) C(15)—C(4)—N(3)—C(2) 148.7(7)C(7)—C(12)—C(11)—C(10) −0.7(6) F(14)—C(10)—C(11)—C(12) −178.7(10)C(9)—C(10)—C(11)—C(12) 2.5(7) C(12)—C(7)—N(6)—C(2) −62.8(7)N(6)—C(7)—C(8)—O(13) −0.5(5) N(6)—C(7)—C(8)—C(9) 178.9(9)C(8)—C(7)—C(12)—C(11) −0.9(6) C(12)—C(11)—C(10)—F(14) −178.7(10)C(12)—C(11)—C(10)—C(9) 2.5(7) O(13)—C(8)—C(7)—C(12) −178.5(10)C(9)—C(8)—C(7)—N(6) 178.9(9) C(7)—C(8)—C(9)—C(10) 0.7(6)C(10)—C(9)—C(8)—O(13) −179.8(9) C(8)—C(9)—C(10)—C(11) −2.5(7)

Intermolecular Non-Bonded Distances (H Omitted)

Minimum distance=1.95 A: Maximum distance=3.50 A

Atom(1)-Atom(2) Distance ns np Ta Tb Tc x(2) y(2) z(2) S(1)—F(14)3.466(4) 3 1 0 1 1 0.43782 1.08343 0.53429 F(14)—C(9) 3.406(8) 3 1 −1 01 −0.48312 0.10096 0.45098 O(16)—O(17) 3.319(6) 4 1 −1 −1 1 −0.599440.68598 0.76806 O(16)—O(13) 2.635(6) 4 1 0 0 1 −0.10236 0.8833 0.86719O(16)—N(6) 2.706(7) 4 1 −1 0 1 −0.74411 1.14494 0.85084 O(16)—N(6)3.207(7) 4 1 0 0 1 0.25589 1.14494 0.85084 O(16)—N(3) 3.434(7) 4 1 −1 01 −0.76558 1.33737 0.79559 O(16)—C(2) 3.471(8) 4 1 −1 0 1 −0.836151.27955 0.83792 O(16)—C(2) 3.399(8) 4 1 0 0 1 0.16385 1.27955 0.83792O(17)—C(5) 3.370(8) 1 1 −1 0 0 −0.99394 1.04756 0.66746 O(17)—N(3)2.732(7) 4 1 −1 0 1 −0.76558 1.33737 0.79559 O(17)—C(4) 3.498(8) 1 1 −10 0 −1.07126 0.97567 0.7166 O(13)—C(5) 3.203(8) 1 1 0 −1 0 0.006060.04756 0.66746 N(3)—C(15) 3.424(8) 4 1 −1 −1 1 −0.7163 0.57321 0.75135S(1)—F(14) 3.466(4) 3 1 0 1 1 0.43782 1.08343 0.53429 F(14)—C(9)3.406(8) 3 1 −1 0 1 −0.48312 0.10096 0.45098 O(16)—O(17) 3.319(6) 4 1 −1−1 1 −0.59944 0.68598 0.76806 O(16)—O(13) 2.635(6) 4 1 0 0 1 −0.102360.8833 0.86719 O(16)—N(6) 2.706(7) 4 1 −1 0 1 −0.74411 1.14494 0.85084O(16)—N(6) 3.207(7) 4 1 0 0 1 0.25589 1.14494 0.85084 O(16)—N(3)3.434(7) 4 1 −1 0 1 −0.76558 1.33737 0.79559 O(16)—C(2) 3.471(8) 4 1 −10 1 −0.83615 1.27955 0.83792 O(16)—C(2) 3.399(8) 4 1 0 0 1 0.163851.27955 0.83792 O(17)—C(5) 3.370(8) 1 1 −1 0 0 −0.99394 1.04756 0.66746O(17)—N(3) 2.732(7) 4 1 −1 0 1 −0.76558 1.33737 0.79559 O(17)—C(4)3.498(8) 1 1 −1 0 0 −1.07126 0.97567 0.7166 O(13)—C(5) 3.203(8) 1 1 0 −10 0.00606 0.04756 0.66746 N(3)—C(15) 3.424(8) 4 1 −1 −1 1 −0.71630.57321 0.75135ns is the symmetry operator number −(* denotes inversion indicator)np is the lattice point numberTa, Tb, & Tc are unit cell translations. The symmetry operations are:

+X, +Y, +Z  1

½−X, −Y, ½+Z  2

½+X, ½−Y, −Z  3

−X, ½+Y, ½−Z  4

Biological Examples

Animals: Experiments were performed on adult male Sprague-Dawley rats(Harlan, Indianapolis, Ind., USA) with implanted jugular vein catheter(JVC), weighing 250-300 grams, housed individually per cage with a 12hour light: 12 hour dark cycle at constant room temperature 21±2° C.Animals had access to food and water ad libitum.

Compounds: Fentanyl citrate (Hospira, Lake Forest, Ill., USA) wasadministered intravenously. The compounds of the invention wereadministrated intravenously.

Nociceptive testing/Thermal sensitivity: Antinociception was assayedusing the tail flick assay (Model IITC 33(T), IITC Life Science,Woodland Hills, Calif., USA), in which a beam of light was focused onthe dorsal surface of the rat tail, approximately 5 cm from the tip ofthe tail. The intensity of the light was adjusted so the baselinelatency was 2-4 seconds. Cutoff time was set to 12 seconds in order toprevent any tissue damage. All testing was conducted by an experimenterwho was unaware of the treatment of an individual rat.

Experimental paradigm A: This study was designed to evaluate the effectof the compounds of the invention on augmentation of fentanyl response.Prior to the beginning of the experiment, base line latency values wereestablished to withdrawal time between 2-3 seconds. Animals received atreatment compound of the invention or the vehicle fifteen minutesbefore the first fentanyl (25 μg/kg, IV) administration, viapre-implanted jugular vein catheter (JVC). Tail flick latency valueswere recorded at 60, 120 and 180 minutes post first fentanyladministration. Second fentanyl administration was administered 210minutes post first fentanyl administration, via JVC route. Tail flicklatency's were recorded at 30, 60, 90, 120 and 150 minutes post secondfentanyl administration.

Experimental paradigm B: This study was design to evaluate the effect ofthe compounds of the invention on augmentation of fentanyl response,mimicking a clinical setting. Prior to the beginning of the experiment,base line latency's were established to withdrawal time between 2-3seconds. Animals received a treatment compound of the invention or thevehicle fifteen minutes prior to the first fentanyl (25 μg/kg, IV)administration, via pre-implanted jugular vein catheter (JVC). Multipledoses of fentanyl (10 μg/kg, IV) were administrated respectively to bothgroups at 75, 135, 195 minutes post first fentanyl administration. Lastfentanyl (25 μg/kg, IV) administration was given right after 240 minpost first fentanyl (25 μg/kg, IV) administration, Tail flick latency'swere recorded at 30, 60, 90, 120, 150, 180, 210, and 240 min post firstfentanyl administration.

Data analysis: Data analysis was preformed using GraphPad Prismsoftware, where latency of withdraw from heat stimuli was comparedbetween the vehicle and treated groups. The data was graphed andanalyzed as percent of Possible Maximum Effect (% MPE=[(postinjectionTFL−baseline TFL)/(12−baseline TFL)×100). Animals whose baseline latency(baseline: latency of withdraw prior to administration of drug orvehicle) withdraw was more than 6 seconds were excluded from the study.Two way analysis of variance (ANOVA), with Benferroni correction formultiple comparisons was used to determine statistical significance(p<0.05) at the different time points.

Analysis of compound activity: Two different methods were utilized toassess activity of the compounds of the invention, and generate SARvalues.

-   -   1. Response Area Analysis: Analysis was based on deriving the        value representing the % of the maximum possible response for        the time periods under question. For example, the maximum        possible value for 5% MPE time-points=5×100=500. For a rat with        maximal possible effect (% MPE) scores of 100, 75, 50, 25 and 10        for the time-points, T30, T60, T90, T120 and T150, the total        score or SUM=260. This value was (260/500)×100=52% of maximum=%        MAX. This value was calculated for every single subject in the        group. Mean and standard error was calculated for the groups,        and one way analysis of variance (ANOVA), with Benferroni        correction for multiple comparisons were used to determine        statistical significance.    -   2. Regression analyses to evaluate time to half maximal        response: To generate time to half maximal response, the data        was converted to % change from the baseline, and time points        were converted to log ten values. Regression analysis was        performed on each subject in the group and time to half maximal        response was generated. The differences between mean values of        the groups was determined by running a one-way analysis of        variance (ANOVA) and using the Error Mean Square term from the        ANOVA, with the Bonferroni correction for multiple comparisons        between means.

Experimental Example 1 Inhibition of Peroxynitrite Oxidation ByCompounds of the Invention

Luminol like compound “L-012” is a chemilumescent probe that isactivated by the presence of a ROS and/or RNS such as peroxynitrite. Inthis assay L-012 was oxidized by the addition of3-Morpholino-sydnonimine (“SIN-1”), a known peroxynitrite generator. Theemitted fluorescence was directly proportional to the concentration ofSIN-1. The compounds of the invention were then assessed for theirability to scavenge peroxynitrite by blocking the increase ofperoxynitrite induced fluorescence. Concentrations of compound producing50% inhibition of the fluorescence signal (IC₅₀ values) were calculatedby testing the compounds of the invention at various concentrations andaveraging the IC₅₀ of three plates.

L-012 Assay Protocol

The following solutions were added to white polystyrene, non-sterile96-well plate (Costar #3912): 196 μL of 50 μM L-012; 2 μL of the testcompound; and 24 μL of 500 mM SIN-1. Fresh stock of 50 μM L-012 in PBSpH 7.4 was made for each 96 well plate that was analyzed. The plateswere read in a FlexStation3 (Molecular Devices) using the luminescentmode at 37° C. for 30 min, with a measurement every 30 seconds and anintegration time of 100 msec.

L-012 Data Collection and Analysis

Luminescence signal was recorded by the FlexStation3 kinetically at 37°C. for 30 minutes, with a measurement every 30 seconds and anintegration time of 100 msec. The data in rows A and H were dropped fromeach plate to exclude the possibility of an ‘edge effect’. Thus, eachtest compound had 3 replicates per plate. The 15 minute data time pointwas plotted to determine % control value or IC50 for follow-upconcentration-response curves (Table 2).

TABLE 2 In vitro Inhibition of SIN-1 Generated Peroxynitrite InducedFluorescence of L-012 by Compounds of the Invention. Average standardCompound IC₅₀ (uM) dev n 2 0.13 0.02 3 26 0.14 0.01 3 21 0.26 0.02 3 10.27 0.01 3 27 0.30 0.03 3 24 0.54 0.12 3 20 0.55 0.08 3

Experimental Example 2 Inhibition of Peroxynitrite Mediated Cytotoxicity

ATPlite is an Adenosine TriPhosphate monitoring system based on fireflyluciferase. ATP monitoring was used as a measure of PC12 cell viability,as it is present in all metabolically active cells and its concentrationvery rapidly declines as cells undergo necrosis and apoptosis. ATPliteis based upon the production of light given off during the reaction ofATP & D-Luciferin in the presence of Luciferase. The emitted light isproportional to the ATP concentration. Compounds of the invention wereassessed for their ability to inhibit SIN-1 induced cell toxicity.Inhibitory concentrations, IC₅₀ values, were calculated by testingcompounds of the invention at various concentrations and averaging theIC₅₀ of two plates.

Cell Preparation

All cell work was carried out under the cell culture hood in a sterileenvironment. On Day 1, rat pheochromocytoma cells (PC12) cells weredissociated from T75 flasks with 0.25% trypsin/EDTA for approximately 2minutes. Cells were harvested by adding 10 mL of F12K media to eachflask and collecting cells into a sterile 15 mL conical tube. From this,10 μL of cells were placed into 90 μL of trypan blue in a 1.5 mLmicrofuge tube. This was equal to a dilution factor of 10. The microfugetube was mixed via vortex briefly and 10 μL was added into one side of ahemacytometer. The cells were counted in the 4 large corner quadrants ofthe hemacytometer under the inverted microscope at 10× and the averageof the 4 quadrants was calculated. Blue cells were not counted as thesecells represented non-viable cells. The number of cells present in 1 mLof media was determined using the following equation: cells/mL=(# cellscounted/# squares counted)×10⁴×dilution factor. (For example: 2.5×10⁶cells/mL=(100 cells/4 squares)×10000×10). Harvested cells were dilutedin F12K media containing high serum (10% horse serum, 5% fetal bovineserum, 1% penicillin streptomycin) so that there were 300,000 cells in 1mL. From this, 100 μL of cells/media were added to a sterile,clear-bottom collagen-coated 96-well plate (30,000 cells/well). Thecells were allowed to attach overnight at 37° C., 5% CO₂.

Inhibition of Peroxynitrite Mediated Cytotoxicity—Protocol

All cell work is carried out under the cell culture hood in a sterileenvironment. On Day 2, media was removed from each plate and replacedwith 100 μl of F12K containing low serum (1% horse serum, 1% penicillinstreptomycin). To test the concentration-response of compounds of theinvention, serial dilutions of these compounds of 5 mM down to 0.02 mMwere prepared in PBS. Each dilution (2 μl volume) was added to theappropriate wells in quadruplicate. Test compounds were incubated on thecells at 37° C. before adding 2 μl of 50 mM SIN-1. The cells were thenincubated overnight at 37° C.

On Day 3, 10.25 ml of ATPlite buffer was added to 1 vial of lyophilizedsubstrate solution (ATPlite 1 step luminescence ATP detection Assaysystem (Perkin Elmer, #6016731)). To each plate, 100 μl/well oflyophilized solution was added and mixed on an orbital plate shaker for2 min at 250 rpm. The plate was read within 5 min the FlexStation3 inluminescence mode.

Cell Based Data Analysis:

Luminescence signal was recorded by the FlexStation3 and percent ofcontrol was calculated. The wells containing only cells were used tocalculate 100% of control, wells containing only SIN-1 was used tocalculate 0%. The remaining data was plotted as % of control bysubtracting the average of the SIN-1 controls and dividing by the 100%control value (Table 3).

TABLE 3 Inhibition of SIN-1 Cytotoxicity Average Standard CompoundIC50(uM) dev n 2 3.7 0.80 2

Experimental Example #3: Preservation of Fentanyl Analgesia in Rat TailFlick Assay

A rat tail flick assay was conducted to assess the effect of compound 2on analgesia induced by fentanyl. The first group of rats was injectedintravenously with a compound and then 15 minutes later was injectedwith fentanyl (25 μg/kg). The second group of rats was injectedintravenously with fentanyl (25 μg/kg) only. At 60, 120 and 180 minutespost-fentanyl administration, tail-flick latency values were obtainedfor both groups and recorded. After a period of 30 minutes (210 totalminutes from the first administration of fentanyl), a second dose offentanyl was given to both groups and at 30, 60, 90, 120 and 180 minutesfollowing the fentanyl injection, tail-flick latency (TFL) was measured.The percentage of the maximal possible effect (% MPE), calculated as[(post-injection TFL−baseline TFL)/(12-baseline TFL)×100] was obtainedFIG. 1.

Experimental Example #4 Preservation of Fentanyl Analgesia in HargreavesThermal Plantar Model

A plantar assay measuring paw withdrawal to a high-intensity thermallight beam, the so-called Hargreaves endpoint (see Hargreaves et al.,1988, Pain 32:77-88), was conducted to assess the effect of compound 2on analgesia induced by fentanyl. The first group of rats was injectedintravenously with compound 2 and then 15 minutes later was injectedwith fentanyl (25 μg/kg). The second group of rats was injectedintravenously with fentanyl (25 μg/kg) only. At 20, 40, 60, 90 and 120minutes post-fentanyl administration, paw withdrawal latency (PWL)values were obtained for both groups and recorded. After a period of 30minutes (150 total minutes from the first administration of fentanyl), asecond dose of fentanyl (25 μg/kg) was given to both groups and PWLvalues were obtained at 20, 40, 60, 90 and 120 minutes post-fentanyladministration. This data is represented graphically in FIG. 2.

Experimental Example #5 Opioid Dose-Sparing

Compound 2 was administered in conjunction with an opioid dosing regimendesigned to simulate patient controlled analgesia wherein small doses ofanalgesic are administered on-demand by patients follow a loading dose.In this experiment, vehicle or compound 2 were given to rats prior tofive doses of fentanyl given hourly as depicted in FIG. 3, and pawwithdrawal latencies values were obtained and used to calculate % MPE.Significant levels of analgesia were maintained in the drug treatedgroup indicating that lower doses were required to maintain analgesia.

Experimental Example #6 Duration of Action of Compound 2

After a single IV dose of compound 2 in a rat, latency values wereobtained at 30, 60, 90, and 120 minutes after each of three doses offentanyl (25 μg/kg) separated by 2.5 hours (FIG. 4).

Experimental Example #7 Duration of Action of Compound 2

After a single IV dose of compound 2 in a rat, latency values wereobtained at 30, 60, 90, and 120 minutes after each of two daily doses offentanyl (25 μg/kg). On the second day, two subsequent daily doses offentanyl were given in an identical fashion as on day 1, but noadditional compound 2 was administered (FIG. 5).

Experimental Example #8 Duration of Action of Compound 2

After a single IV dose of compound 2 in a rat, latency values wereobtained at 20, 40, 60, 90, and 120 minutes after doses of fentanyl (25μg/kg) given daily for four days (FIG. 6).

Experimental Example #9 Effects of Compound 2 on Existing OpioidTolerance/Hyperalgesia

Rats were treated with three doses of fentanyl for two days as depictedin FIG. 7. On day 3, vehicle or a single IV dose of compound 2 was givento a rat after fentanyl challenge and latency values were obtained at20, 40, 60, 90, and 120 minutes. This data shows that compounds of theinvention can reverse pre-established opioid tolerance/hyperalgesia.

Experimental Example #10 Effect of Compound 2 on Capsaicin-InducedHyperlagesia

Rats were given compound 2 IV prior to an intra-plantar administrationof capsaicin, and withdrawal latencies were measured. This data,depicted graphically in FIG. 8, shows that compounds of the inventioncan prevent the development of hyperalgesic pain states produced byactivation of primary afferent sensory neurons that express the TRPV1receptor/channel.

Experimental Example #11 Effect of Compound 2 on Incisional Hyperlagesia

Vehicle, a reference compound (indomethacin), or compound 2 was given IVat the conclusion of a surgical incision and latency values weremeasured 24 hours later (0 timepoint) and 30 and 60 minutes thereafter.Determination of latencies was subsequently conducted 48 hours and 72hours in the same manner (FIG. 9).

Experimental Example #12 Effect of Compound 2 on Established IncisionalHyperlagesia

Vehicle, a reference compound (indomethacin), or compound 2 was given 24hours after the conclusion of a surgical incision; latency values weremeasured 24 and 48 hours later (0 timepoint) and 30 and 60 minutesthereafter (FIG. 10). This data demonstrates that compounds of theinvention can rereverse hyperalgeisa due to post-surgical pain.

Experimental Example #13 Compound 2 Inhibits Hyperalgesia Caused bySIN-1

Vehicle or compound 2 was given IV 15 minutes before the firstadministration of SIN-1 (1 mg/kg). Latency was measured for the firsttime 2 hours later (Testing 1). SIN-1 (1 mg/kg) was again administered30 minutes after Testing 1 and latency was again measured 2 hours later(Testing 2). SIN-1 (1 mg/kg) was again administered 30 minutes afterTesting 2 and latency was again measured 2 hours later (Testing 3). Theresults, depicted in FIG. 11, demonstrate that compounds of theinvention can block the development of hyperalgesia caused by SIN-1.

Experimental Example #14 Effect of Compound 2 on Opioid-Induced Toxicity

In this study, the effect of a single dose of Compound 2, administeredpreemptively, on fentanyl induced toxicity (death) and analgesia wasevaluated. The negative control was vehicle (0.5% methyl cellulose inphosphate buffered saline). Male Sprague-Dawley Rats (Harlan, Ill)250-300g at time of dosing were used.

Paw withdrawal latency to a thermal stimulus was assessed using aradiant heat source (Ugo Basile, Italy) aimed at the plantar surface ofthe left hind paw (Hargreaves test; Hargreaves et al., 1988, Pain32:77-88). A cut-off latency of 40 seconds was set to avoid tissuedamage. Baseline latencies were taken prior to drug administrations, andCompound 2 (25 mg/kg, i.v.) or vehicle was administered. Fifteen minuteslater, the first dose of fentanyl was administered (25 μg/kg, i.v.),behavior was subsequently assessed 15 and 45 minutes post-fentanyl. Fiveadditional fentanyl doses were administered at 60 minute intervals withbehavioral assessment 15 and 45 minutes after each dose.

Statistical significance was determined on untransformed data using atwo-way analysis of variance (Graphpad Prism, NC). Significant maineffects were analyzed further by subsequent Bonferronni post-hoc test.The level of significance was set at p<0.05. Data are shown asmean±S.E.M. (with an asterisk to denote significance as compared tovehicle treated controls). The percent survival was calculated accordingto the following equation;

% survival=[(initial n−expired n)/(initial n)]×100

Table 3 summarizes in vivo pharmacology data. Table 4 summarizes pawwithdrawal latency data, illustrated in FIG. 17. Table 5 summarizessurvival (%), illustrated in FIG. 16.

TABLE 3 Method and Gender Species/ Time of Doses and No. Noteworthy TestSystem Strain Administration (units) per Group Findings Fentanyl Rat/SDi.v. Compound 2 single M, Compound 2 induced 25 mg/kg n = 6significantly toxicity (preemptively) (initial) reduced death (Survival)Fentanyl 25 μg/kg attributable to (hourly for 6 h) fentanyl FentanylRat/SD i.v. Compound 2 single M, Compound 2 induced 25 mg/kg n = 6significantly toxicity (preemptively) (initial) preserved (Hargreaves)Fentanyl 25 μg/kg analgesic (hourly for 6 h) efficacy of fentanyl

TABLE 4 Paw withdrawal latency. vehicle vehicle vehicle vehicle vehiclevehicle Cpd 2 Cpd 2 Cpd 2 Cpd 2 Cpd 2 Cpd 2 baseline 20.12 16.64 21.7 1920.1 17.98 19.3 17.01 21.3 20.3 17.6 19.03 15 40 40 40 40 40 40 40 40 4040 40 40 45 37.8 40 40 38.9 37.2 40 40 39.2 40 37.8 39 40 75 40 40 40 4040 40 40 40 40 40 40 40 105 25.34 21.78 24 23.61 19.67 21.23 40 40 37.5238 35.96 135 40 40 40 40 40 40 40 40 40 40 40 165 18.26 15.33 22.22 4035.63 34.68 40 35.33 195 40 40 40 40 40 40 40 40 225 16.89 11.3 33.5436.45 33.98 40 25.34 255 40 40 40 40 40 40 40 285 6.78 15.55 22.81 26.1438.02 18.61 25.59 315 33.65 40 40 40 40 40 40 345 7.22 25.33 24.78 36.9920.78 25.88

TABLE 5 Survival (%). vehicle cpd 0 h 100 100 1 h 100 100 2 h 100 100 3h 50 83.33 4 h 33.3 83.33 5 h 33.3 83.33 6 h 16.66 83.33

Over the time course of the experiment animals died due to respiratorydepression or other acute toxicities associated with repeatedadministration of high doses of fentanyl. Compound 2 reduced the extentof death such that a significant increase in % survival was noted (FIG.16): 1/6 animals died in the “Compound 2” group and 5/6 animals died inthe vehicle treated group (the remaining animal was sacrificedmoribund). This suggests Compound 2 reduces opioid induced toxicity andimproves safety.

For the analgesia measurements, animals were baselined and Compound 2(25 mg/kg, i.v.) was administered as a single dose 15 min prior tofentanyl (25 μg/kg, i.v.). Additional fentanyl doses were administeredat 60 min intervals and behavior assessed 15 and 45 minutes after eachfentanyl administration (FIG. 17).

In vehicle treated animals, fentanyl produced robust analgesia 15minutes after each dose, but paw withdrawal latencies returned to nearbaseline levels by 45 minutes post-dose. Paw withdrawal latencies fellbeneath baseline levels 45 minutes after the 4^(th), 5^(th) and 6^(th)doses of fentanyl, consistent with opioid induced hyperalgesia.

Compound 2 produced a statistically significant preservation ofanalgesic efficacy 45 minutes after dosing with fentanyl. Paw withdrawallatencies did not fall beneath baseline levels 45 minutes after the4^(th), 5^(th) and 6^(th) doses of fentanyl. This suggests pretreatmentwith Compound 2 prevented development of opioid induced hyperalgesia(FIG. 16).

Preemptively administered Compound 2 significantly reduced deathsattributable to fentanyl. Additionally, Compound 2 (25 mg/kg, i.v.)preserved the analgesic efficacy of fentanyl and prevented theoccurrence of opioid induced hyperalgesia. This data suggests thatCompound 2 reduces opioid toxicity, sustains opioid analgesic efficacyand prevents opioid induced hyperalgesia. In clinical settings, Compound2 may improve the safety profile of opioids, while simultaneouslyaugmenting analgesia.

Experimental Example #15 Effect of Compound 2 on Thermal Hyperalgesia,Tactile Allodynia and Edema Induced by Intraplantar Freund's CompleteAdjuvant

Compound 2 was evaluated in the rat Freund's complete adjuvant (FCA)model of inflammatory pain. The rat Freund's complete adjuvant (FCA)intraplantar model of inflammatory pain produces behaviors similar tothose observed in patients and has been widely used to assess novelpharmacological treatments (Lam et al., 2008, J Ethnopharmacol.120:44-50).

The positive control was celecoxib (TRC, Montreal), and the negativecontrol was vehicle (0.5% methyl cellulose, in phosphate bufferedsaline). Male Sprague-Dawley Rats (Harlan, ID 250-300g at time of dosingwere used. The Hargreaves apparatus was obtained from Ugo Basile, Italy.The Freund's complete adjuvant was obtained from Sigma, St. Louis, andthe electronic von frey was obtained from Stoelting, Ill.

For this assay, paw withdrawal latency to a thermal stimulus wasassessed using a radiant heat source (Ugo Basile, Italy) aimed at theplantar surface of the left hind paw (Hargreaves test; Hargreaves etal., 1988, Pain 32:77-88). A cut-off latency of 40 seconds was set toavoid tissue damage. Paw withdrawal thresholds to a non-noxious tactilestimulus was assessed using an electronic von Frey apparatus thatpresented an increasing mechanical force to the plantar surface of thehind paw. Paw volume was measured by displacement of water and assuminga tissue density equal to that of water. Baseline latencies, thresholdsand volume were taken prior to FCA injection and re-assessed 24-72 hourslater. Compound 2 or vehicle was administered (25 mg/kg, i.v.; or 3-100mg/kg, p.o.) either once immediately prior to FCA (preemptive) or 24-72hours post-FCA (curative). When Compound 2 was administeredpreemptively, behavior or paw volume was assessed 24-72 hourspost-dosing. When Compound 2 was administered curatively, behavior orpaw volume was assessed 30-180 min post-dosing. Celecoxib (30 mg/kgp.o.) served as the positive control.

Intraplantar injection was performed under 2.5-4% isoflurane/O₂anesthesia, delivered via nose cone. After induction of anesthesia, theinjection site was prepared in a sterile manner and 100 μl of a 50%suspension of FCA injected. After injection, animals were weighed andallowed to recover before being returned to their home cages.

Statistical significance was determined on untransformed data using atwo-way analysis of variance (Graphpad Prism, NC). Significant maineffects were analyzed further by subsequent Bonferronni post-hoc test.The level of significance was set at p<0.05. Data are shown asmean±S.E.M. (with an asterisk to denote significance as compared tovehicle treated controls).

Intraplantar injection of 100 μl of FCA into the hind paw resulted inthe development of thermal hyperalgesia, tactile allodynia (as assessedby electronic von Frey) and edema as indicated by a decreased latency toa noxious thermal stimulus, decreased threshold to a non-noxious tactilestimulus and increase in paw volume (FIGS. 18-20). Compound 2 (25 mg/kg,i.v.) administered preemptively prevented the development of thermalhyperalgesia (FIG. 18A) and tactile allodynia (FIG. 18B). Statisticalsignificance, as compared to vehicle treated controls, was reached 72hours post-FCA for thermal hyperalgesia (FIG. 18A) and 24, 48 and 72hours post-FCA for tactile allodynia (FIG. 18B).

Table 6 summarizes the in vivo pharmacology data. Table 7 summarizes pawwithdrawal latency (s), illustrated in FIG. 18A. Table 8 summarizes pawwithdrawal latency (s), illustrated in FIG. 18B. Table 9 summarizes pawwithdrawal threshold (g), illustrated in FIG. 19A. Table 10 summarizespaw volume (mL), illustrated in FIG. 20A. Table 11 summarizes paw volume(mL), illustrated in FIG. 20B.

TABLE 6 In vivo pharmacology data. Method and Gender Species/ Time ofDoses and No. Noteworthy Test System Strain Admin. (units) per GroupFindings FCA induced Rat/SD i.v. Single dose M, n = 6 Compound 2 thermal25 mg/kg significantly prevented hyperalgesia (preemptive) FCA-thermaland tactile hyperalgesia and allodynia tactile allodynia FCA inducedRat/SD p.o. Single dose M, n = 6 Compound 2 tactile allodynia 25 mg/kgsignificantly prevented (preemptive) FCA-induced tactile allodynia;effect lasted for 3 days and was not reproduced by celecoxib FCA inducedRat/SD p.o. 3, 10, 30 and 100 M, n = 6 Compound 2 thermal tactile mg/kg(curative) significantly reversed allodynia FCA-induced tactileallodynia FCA induced Rat/SD p.o. 10 and 30 mg/kg M, n = 6 Compound 2did not edema (preemptive and significantly reverse curative)FCA-induced edema

TABLE 7 Paw withdrawal latency (s), illustrated in FIG. 18A. baselineday 1 day 3 day 4 vehicle 18.72 16 12.7 10.66 vehicle 22.5 12.65 9.769.28 vehicle 21.2 12.2 9.16 9.85 vehicle 21.55 8.72 8.84 9.5 vehicle19.45 26.63 11.3 7.73 vehicle 20.52 23.6 16.5 13.85 compound 2 16.6 14.412.5 14 compound 2 17.55 156.3 19.12 16.25 compound 2 20.74 15.1 19.516.51 compound 2 20.9 15.1 19.5 16.51 compound 2 20.6 15.7 15.7 16.6compound 2 19.33 22.7 17.7 18.2

TABLE 8 Paw withdrawal latency (s), illustrated in FIG. 18B. baselineday 1 day 3 day 4 vehicle 33.2 15.5 13.8 13.65 vehicle 32.8 14 17.5 9.8vehicle 29.6 17.6 15.9 18.2 vehicle 31.3 15.1 20.2 12.8 vehicle 32.229.4 28.5 24.7 vehicle 28.1 12.4 15.4 15.65 compound 2 29.6 36.7 29.8 25compound 2 32.5 26.7 28 27.23 compound 2 29.8 38.9 30 36.5 compound 227.5 25 27.7 31.3 compound 2 31.4 27 23.5 31.9 compound 2 29.6 29 33.824.3

TABLE 9 Paw withdrawl threshold (g), illustrated in FIG. 19. baselineBL(D1) 30 60 vehicle 38.27 17.2 13.1 15.2 vehicle 31.8 15.9 16.3 15.7vehicle 37 20.1 11.3 13.4 vehicle 38.13 6.4 9.6 8.9 vehicle 31.1 10.113.8 11.2 vehicle 36.1 18.3 16.7 17.5 vehicle 35.4 22.5 19 20.1 vehicle30.9 17.6 16.8 17 vehicle 33.4 21.4 20 19.5 vehicle 37.3 19.2 15 16.4vehicle 30.4 10.6 16.8 15.6 Compound 2 36.1 20.5 22.1 22 (3 mg/kg)Compound 2 32.4 15.3 16.9 16.5 (3 mg/kg) Compound 2 35.8 18.5 21 21.4 (3mg/kg) Compound 2 33.4 16.5 12.6 15.7 (3 mg/kg) Compound 2 34.4 17.327.2 22.3 (3 mg/kg) Compound 2 30.9 15.8 17.5 18 (3 mg/kg) Compound 228.6 8.9 17 28.2 (10 mg/kg) Compound 2 26.67 12.3 22.2 25.5 (10 mg/kg)Compound 2 37.9 12.3 22.2 25.5 (10 mg/kg) Compound 2 31.3 8.1 12.6 12.6(10 mg/kg) Compound 2 39 22.5 31.6 36.3 (10 mg/kg) Compound 2 31.8 15.125 30.9 (10 mg/kg) Compound 2 33.2 15 19.3 24 (30 mg/kg) Compound 2 34.919.2 32.6 24.2 (30 mg/kg) Compound 2 36.6 18.2 27.3 26.5 (30 mg/kg)Compound 2 35.6 22.5 30.2 21.6 (30 mg/kg) Compound 2 34.9 17.8 26.3 27.4(30 mg/kg) Compound 2 33.4 16.5 19.5 31.1 (30 mg/kg) Compound 2 31.812.8 28.6 31.2 (100 mg/kg) Compound 2 26.6 13.9 27.9 27.5 (100 mg/kg)Compound 2 27.6 16 19.3 35.3 (100 mg/kg) Compound 2 33.33 19.3 34.6 29.1(100 mg/kg) Compound 2 37.8 23.51 31.6 30.3 (100 mg/kg) Compound 2 31.912.6 21 34.6 (100 mg/kg) celecoxib 31.8 9.6 19.5 29.9 (30 mg/kg)celecoxib 39.4 33.6 24 32.4 (30 mg/kg) celecoxib 31.8 11.6 11.3 19.8 (30mg/kg) celecoxib 25.93 7.4 18.3 23.2 (30 mg/kg) celecoxib 36.6 13.6 16.330.9 (30 mg/kg) celecoxib 37.73 13.6 32.1 29.9 (30 mg/kg)

TABLE 10 Paw volume (mL) illustrated in FIG. 20A. FCA/ FCA/ FCA/ FCA/FCA/ FCA/ Vehicle Vehicle Vehicle Vehicle Vehicle Vehicle Pre-FCA 1.81.7 1.8 1.7 1.7 1.9 24 hrs pos 2.8 3 3.1 3 3.1 3.1 48 hrs pos 2.9 2.9 33 3.1 3.1 compound 2 (10 mg/kg) 1.7 1.6 1.8 1.9 1.8 1.7 2.7 2.6 3.1 3.13.1 2.7 2.6 2.5 2.9 2.9 3.2 2.6 compound 2 (30 mg/kg) 1.6 1.7 1.9 1.81.7 1.8 2.1 2.9 3 3 2.9 2.9 2.5 2.8 3.1 2.8 2.7 2.9 Celecoxib CelecoxibCelecoxib Celecoxib Celecoxib Celecoxib 1.8 1.9 1.9 1.7 1.5 1.7 2.8 3.12.7 3 2.2 2.7 2.7 2.7 2.8 2.8 2.2 2.8

TABLE 11 Paw volume (mL) illustrated in FIG. 20B. FCA/Vehicle FCA/ FCA/FCA/ FCA/ FCA/ FCA/ FCA/ FCA/ Vehicle Vehicle Vehicle Vehicle VehicleVehicle Vehicle Vehicle Pre-FCA 1.8 1.8 1.6 1.6 1.9 1.7 1.7 1.8 Pre-Drug3.1 2.9 2.8 2.6 3.1 2.9 2.8 3.2 1 hr 3.1 2.8 2.8 2.6 3 2.9 2.7 3.2 3 hrs3 3 2.9 2.6 3 3 2.8 3.3 Pre-Drug (D2) 2.9 3.1 2.9 2.4 3 2.5 2.8 3 1 hr(D2) 2.9 3.1 2.9 2.5 2.9 2.7 2.8 3 3 hrs (D2) 2.8 3.1 2.8 2.6 3 2.6 2.82.9 FCA/Compound 2, 10 mg/kg FCA/GAL- FCA/GAL- FCA/GAL- FCA/GAL-FCA/GAL- FCA/GAL- FCA/GAL- FCA/GAL- Pre-FCA 1.7 1.6 1.8 1.6 1.8 1.8 1.51.7 Pre-Drug 3.1 2.8 3.1 2.8 2.9 3 2.7 3.4 1 hr 2.9 2.9 3.1 2.7 2.8 32.7 3.1 3 hrs 2.9 2.9 3.1 2.7 2.9 2.9 2.4 3.2 Pre-Drug (D2) 2.7 2.7 32.6 2.6 2.8 2.4 3.2 1 hr (D2) 2.7 2.7 2.9 2.7 2.5 2.8 2.3 3.2 3 hrs (D2)2.7 2.7 3 2.6 2.5 2.8 2.4 3.2 FCA/Compound 2, 30 mg/kg FCA/GAL- FCA/GAL-FCA/GAL- FCA/GAL- FCA/GAL- FCA/GAL- FCA/GAL- FCA/GAL- Pre-FCA 1.7 1.81.5 1.8 1.7 1.7 1.9 1.7 Pre-Drug 2.8 3 2.9 3.2 3 2.6 3.4 2.8 1 hr 2.6 32.8 2.8 2.9 2.5 3.1 2.7 3 hrs 2.8 2.9 2.7 3 2.8 2.6 3.2 2.7 Pre-Drug(D2) 2.4 2.7 2.6 2.8 2.9 2.4 3.1 2.7 1 hr (D2) 2.5 2.7 2.6 2.9 2.7 2.43.2 2.7 3 hrs (D2) 2.5 2.7 2.6 2.8 2.9 2.4 3 2.7 FCA/celecoxib, 30 mg/kgFCA/ FCA/ FCA/ FCA/ FCA/ FCA/ FCA/ FCA/ Celecoxib Celecoxib CelecoxibCelecoxib Celecoxib Celecoxib Celecoxib Celecoxib Pre-FCA 1.7 1.7 1.71.8 1.7 1.9 1.7 1.7 Pre-Drug 2.8 3.1 2.8 2.9 3.1 3 3.2 2.8 1 hr 2.7 2.92.6 2.7 3.1 3 3 2.7 3 hrs 2.7 2.9 2.7 2.8 3.1 2.7 2.9 2.6 Pre-Drug (D2)2.4 2.6 2.3 2.5 2.6 2.7 2.8 2.3 1 hr (D2) 2.3 2.6 2.3 2.4 2.7 2.5 2.72.4 3 hrs (D2) 2.4 2.5 2.3 2.5 2.7 2.6 2.8 2.3

When Compound 2 (3-100 mg/kg, p.o.) was administered 24 hours post-FCA,a significant reversal of FCA induced tactile allodynia was noted 30 and60 minutes post-administration of 10, 30 and 100 mg/kg (FIG. 19). Thepositive control, celecoxib (30 mg/kg, p.o.) also produced astatistically significant reversal 60 minutes post-administration (FIG.19).

Compound 2 (10 or 30 mg/kg, p.o.) did not have a statisticallysignificant effect on FCA induced edema as measured by increase in pawvolume, regardless of whether administered preemptively (FIG. 20A) or 24hours post-FCA (FIG. 20B). In contrast, the positive control, celecoxib(30 mg/kg, p.o.), produced a statistically significant block of edema 24and 48 hours post-administration (FIG. 20A and FIG. 20B).

These results suggest that Compound 2, by the i.v. or p.o. route ofadministration, may fully or partially prevent development of FCAinduced tactile allodynia and thermal hyperalgesia. Importantly, in thecase of tactile allodynia, this effect was maintained for at least 3days. This effect was not seen with the same p.o. dose of celecoxib.Furthermore, Compound 2 was also able to reverse establishedinflammatory tactile allodynia when administered p.o., in this caseproducing an effect comparable to celecoxib. Finally Compound 2 did notaffect edema in this model indicating that the effect on pain behavioris not secondary to an anti-inflammatory effect. The action of thecelecoxib control was associated with reductions in edema. Takentogether, these data suggest a clear differentiation between GAL-044 andcelecoxib with respect to mechanism of action and therapeutic utility.

Experimental Example #16 Effect of Compound 2 on Thermal Hyperalgesiaand Edema Induced by Intraplantar Carrageenen

The effect of Compound 2 in the rat carrageenan model of acuteinflammatory pain was evaluated. The intraplantar carrageenan model ofacute inflammatory pain in the rat results in behaviors similar to thatobserved in patients and has been widely used to assess novelpharmacological treatments (Whiteside et al., 2005, J. Pharmacol. Exp.Ther. 314:1234-1240). The objective of this study was to evaluate theability of Compound 2 to reverse or prevent development of thermalhyperalgesia and edema in the rat carrageenan model of acuteinflammatory pain.

The positive control was indomethacin (Sigma, St. Louis), and thenegative control was vehicle (0.5% methyl cellulose). MaleSprague-Dawley Rats (Harlan, Il) were 250-300g at time of dosing. TheHargreaves apparatus was obtained from Ugo Basile, Italy, and thelamba-carrageenan was obtained from Sigma, St. Louis.

For this assay, paw withdrawal latency to a thermal stimulus wasassessed using a radiant heat source (Ugo Basile, Italy) aimed at theplantar surface of the left hind paw (Hargreaves test). A cut-offlatency of 40 seconds is set to avoid tissue damage. Paw volume wasmeasured by displacement of water and assuming a tissue density equal tothat of water. Baseline latencies were taken prior to carrageenanadministration and re-assessed 4 h later. Compound 2 or vehicle wasadministered (10 and 30 mg/kg, p.o.) 3 hrs after carrageenan (curative)and behavior assessed 1 hr later. Additionally, Compound 2 wasadministered 15 minutes prior to carrageenan (preemptive) and behaviorassessed 4 hours post carrageenan. Indomethacin (positive control, 30mg/kg p.o.) was administered prior to carrageenan and behavior assessed4 hrs post-carrageenan.

Intraplantar injection was performed under 2.5-4% isoflurane/O₂anesthesia, delivered via nose cone. After induction of anesthesia, theinjection site was prepared in a sterile manner and 50 μl of a 2%Jamba-carrageenan was injected. After injection, animals were weighedand allowed to recover before being returned to their home cages.

Statistical significance was determined on untransformed data using atwo-way analysis of variance (Graphpad Prism, NC). Significant maineffects were analyzed further by subsequent Bonferronni post-hoc test.The level of significance was set at p<0.05. Data are shown asmean±S.E.M. (with an asterisk to denote significance as compared tovehicle treated controls).

Table 12 summarizes in vivo pharmacology data. Table 13 summarizes pawwithdrawal latency (s) for FIG. 21A. Table 14 summarizes paw volume (mL)for FIG. 21B. Table 15 summarizes paw withdrawal latency (s) for FIG.22A. Table 16 summarizes paw volume (mL) for FIG. 22B.

TABLE 12 In vivo pharmacology. Method and Gender Species/ Time of Dosesand No. Noteworthy Test System Strain Administration (units) per GroupFindings Carrageenan- Rat/SD p.o. 30 mg/kg M, Compound 2 induced(curative) n = 7-8 significantly edema reversed carrageenan- inducededema Carrageenan- Rat/SD p.o. 10 and 30 M, Compound 2 induced mg/kg n =6-7 significantly thermal (curative) reversed hyperalgesia/ carrageenan-edema induced edema but not thermal hyperalgesia Carrageenan- Rat/SDp.o. 10 and 30 M, Compound 2 did induced mg/kg n = 6 not significantlythermal (preemptive) reverse hyperalgesia/ carrageenan- edema inducedthermal hyperalgesia or edema

TABLE 13 Paw withdrawal latency (s) illustrated in FIG. 21A. Cg/ Cg/ Cg/Cg/ Cg/ Cg/ Cg/ Vehicle Vehicle Vehicle Vehicle Vehicle Vehicle VehiclePre- 18.77 17.98 20.25 34.15 14.14 13.02 28.61 Cg 1 hr 8.34 6.27 4.786.84 5.82 3.48 4.07 post- drug compound 2 (10 mg/kg) 19.8 15.34 22.0717.39 31.03 14.07 11.14 3.17 5.54 5.86 8.02 5.73 compound 2 (30 mg/kg)23.87 12.41 16.08 28.86 18.1 22.77 17.58 7.9 5.16 6.78 10.22 16.05 7.567.34

TABLE 14 Paw volume (mL) illustrated in FIG. 21B. Cg/ Cg/ Cg/ Cg/ Cg/Cg/ Cg/ Vehicle Vehicle Vehicle Vehicle Vehicle Vehicle Vehicle Pre- 1.71.7 1.8 1.7 1.8 1.7 1.7 Cg 1 hr 2.5 3 3.2 2.6 2.8 2.8 3.1 post- drugcompound 2 (10 mg/kg) 1.7 1.8 1.8 1.7 1.6 1.8 2.6 2.6 2.9 2.5 2.7 2.7compound 2 (30 mg/kg) 1.6 1.7 1.6 1.7 1.8 1.5 1.6 2.4 2.6 2.6 2.6 2.52.9 2.3

TABLE 15 Paw withdrawal latency (s) illustrated in FIG. 22 A. Cg/ Cg/Cg/ Cg/ Cg/ Cg/ Vehicle Vehicle Vehicle Vehicle Vehicle Vehicle 25.8 4031.89 18.4 15.8 22.79 7.55 13.11 7.25 5.75 5.29 12.85 compound 2 (10mg/kg) 20.94 40 25.09 26.96 13.66 23.25 8.1 5.07 20.71 12.18 11.94 9.38compound 2 (30 mg/kg) 27.75 15.78 35.57 23.26 18.37 25.78 11.57 16.636.81 11 32.38 11.6 Indo Indo Indo Indo Indo Indo 20 40 17 32 23 24 17 3526 28 40 40

TABLE 16 Paw volume (mL) illustrated in FIG. 22B. Cg/ Cg/ Cg/ Cg/ Cg/Cg/ Vehicle Vehicle Vehicle Vehicle Vehicle Vehicle Pre-Cg 1.8 1.6 1.81.8 1.7 1.7 4 hr 2.8 3.1 2.8 2.8 3.2 2.7 post- drug compound 2 (10mg/kg) 1.7 1.6 1.5 1.6 1.7 1.6 3.3 2.8 2.8 3.2 3.1 2.9 compound 2 (30mg/kg) 1.7 1.7 1.6 1.8 1.6 1.7 2.9 2.7 2.7 2.7 2.8 2.7 Indo Indo IndoIndo Indo Indo 1.7 1.5 1.7 1.8 1.5 1.4 2.5 2.4 2.3 2.2 2.3 1.8

Intraplantar injection of 50 μl of carrageenan into the hind pawresulted in the development of thermal hyperalgesia and edema asindicated by a decreased latency to a noxious thermal stimulus andincrease in paw volume. Compound 2 (30 mg/kg, p.o.) significantlyreversed edema when given as a single administration 3 hourspost-carrageenan without significantly reversing thermal hyperalgesia(FIG. 21). When administered preemptively Compound 2 (10 and 30 mg/kg,p.o.) did not significantly prevent either carrageenan-induced edema orthermal hyperalgesia (FIG. 22). When dosed preemptively indomethacin (30mg/kg p.o.), the positive control, produced a statistically significantprevention of both thermal hyperalgesia and edema (FIG. 22).

Compound 2 (30 mg/kg, p.o.) significantly reversed edema when given as asingle administration 3 hours post-carrageenan but did not significantlyreverse thermal hyperalgesia. When administered preemptively, a singledose of Compound 2 (10 and 30 mg/kg, p.o.) did not significantly preventdevelopment of either carrageenan-induced edema or thermal hyperalgesia.These data suggested that preemptive Compound 2 does not prevent thermalhyperalgesia or edema caused by acute inflammation. However, whenadministered curatively reduces edema without significantly reducingthermal hyperalgesia.

Experimental Example #17 Effect of Compound 2 on Tactile AllodyniaInduced by Spinal Nerve Ligation

Compound 2 was evaluated in the rat spinal nerve ligation (SNL) model(Chung model) of neuropathic pain. The spinal nerve ligation (SNL) modelof neuropathic pain in the rat results in behaviors similar to thatobserved in patients (Kim and Chung, 1992, Pain 50(3):355-63) and hasbeen widely used to assess novel pharmacological treatments (Sindrup andJensen, 1999, Pain 83(3):389-400). Compound 2 was evaluated for itsability to reverse or prevent development of tactile allodynia in a ratmodel of neuropathic pain.

The positive control was gabapentin (Toronto Research Chemicals,Canada). The negative control was vehicle (0.5% methyl cellulose;2-hydroxy-propyl-beta-cyclodextrin). Male Sprague-Dawley Rats (Harlan,Il) were 250-300 g at time of dosing. Von Frey filaments were obtainedfrom Stoelting, Ill, and 7-0 silk, 4-0 vicryl sutures were obtained fromEthicon, NJ

The surgical procedure was performed under 2.5-4% isoflurane/O₂anesthesia, delivered via nose cone, and anesthesia was maintained forthe duration of the surgery. After induction of anesthesia, the incisionsite was shaved and prepared in a sterile manner. A midline incision wasperformed, the L5 transverse process removed and the L5 spinal nervestightly ligated with 7-0 silk suture material. The wound was closed inlayers with 4-0 vicryl. Sham-operated control rats underwent identicalprocedures, however, the spinal nerve was not manipulated or ligated.After surgery, animals were weighed and allowed to recover before beingreturned to their home cages.

The effect of Compound 2 on nerve injury induced tactile allodynia wasinvestigated using von Frey filaments 1-3 weeks after tight ligation ofthe L5 spinal nerve. Tactile thresholds were assessed using a series ofcalibrated von Frey monofilaments (Stoelting, Wood Dale, Ill.).Assessment of tactile allodynia was measured as the hind paw-withdrawalthreshold that produced a 50% likelihood of a withdrawal using theup-down method. Thresholds were evaluated before surgery, and they werereassessed 1-3 weeks after SNL surgery. Rats were administered either asingle acute dose of Compound 2 (30 mg/kg) on the day of surgery, QD (10and 50 mg/kg) on days 1-5 post-surgery or QD (30 mg/kg) on days 1-5post-surgery. Tactile thresholds were again assessed either 1 and 3 hrs,3 and 5 hrs or once weekly after administration. Vehicle treated animalswere included and gabapentin (100 mg/kg, i.p.) was used as the positivecontrol. The number of animals per group was 8.

Statistical significance was determined on untransformed data using atwo-way analysis of variance (Graphpad Prism, NC). Significant maineffects were analyzed further by subsequent Bonferronni post-hoc test.The level of significance was set at p<0.05. Data are shown asmean±S.E.M.

Table 17 summarizes in vivo pharmacology data. Table 18 summarizes pawwithdrawal threshold (g) illustrated in FIG. 23. Table 19 summarizes pawwithdrawal threshold (g) illustrated in FIG. 24. Table 20 summarizes pawthreshold (g) illustrated in FIG. 25. Table 21 summarizes paw threshold(g) illustrated in FIG. 26.

TABLE 17 In vivo pharmacology data. Method and Gender Species/ Time ofDoses and No. Noteworthy Test System Strain Administration (units) perGroup Findings SNL-induced Rat/SD p.o. 10 and M, n = 8 Compound 2 didnot tactile 50 mg/kg significantly reverse allodynia SNL-induced tactileallodynia SNL-induced Rat/SD p.o. 50 mg/kg M, n = 8 Compound 2 tactile(fasted) marginally (but allodynia significantly) reversed SNL- inducedtactile allodynia on first day of dosing: gabapentin produced asignificant reversal SNL-induced Rat/SD p.o. 30 mg/kg M, n = 8 Compound2 did not tactile significantly affect allodynia development ofSNL-induced tactile allodynia when administered once on the day ofsurgery SNL-induced Rat/SD p.o. 30 mg/kg M, n = 8 On week 1 aftertactile surgery, Compound allodynia 2 significantly reduced thedevelopment of SNL-induced tactile allodynia when administered qd ondays 1-5 post- surgery

TABLE 18 Paw withdrawal threshold (g) illustrated in FIG. 23. Ve- Ve-Ve- Ve- hicle hicle hicle Vehicle Vehicle Vehicle Vehicle hicle Pre- 1515 15 15 15 15 15 15 Sx Pre- 3.13 2.81 3.13 1.56 4.25 3.73 5.04 3.67Drug 1 hr 2 5.04 10.04 2.38 7.94 4.25 4.25 2.81 (D1) 3 hrs 1.56 3.6710.04 2 3.58 3.73 2.81 2.81 (D1) 1 hr 1.4 1.12 5.04 3.55 2.37 2.2 2.811.56 (D2) 3 hrs 1.85 5.54 3.13 2.81 3.33 3.13 4.25 1.65 (D2) 1 hr 1.42.64 4.25 1.19 4.25 1.58 3.58 1.85 (D3) 3 hrs 1.85 6.66 3.58 2.2 2.372.2 2.37 2 (D3) compound 2 (10 mg/kg) 11.69 15 15 15 15 15 15 15 2.23.13 4.25 3.58 3.73 4.4 2.64 3.13 1.56 4.98 4.25 15 4.25 3.13 2.81 4.4 27.94 1.4 15 2.81 2.64 1.85 3.57 1.31 1.85 1.85 2.38 1.85 2.64 1.65 1.40.4 1.56 1.56 11.69 2.81 2.37 2 3.71 0.44 3.67 1.85 7.94 3.33 2 2.81 4.40.99 2.37 1.56 2.38 2.81 0.65 3.33 2.81 compound 2 (50 mg/kg) 15 15 1515 15 15 15 15 2.81 3.33 3.73 2.81 2.2 5.04 4.25 3.58 2.81 6.64 2.815.56 3.67 5.56 4.23 4.98 1.18 3.13 3.33 6.66 6.64 6.66 3.13 4.7 1.192.81 3.12 7.94 2.81 6.66 1.18 2.37 1.65 4.47 2.81 3.58 2.65 6.58 2.813.33 0.78 6.58 3.67 4.72 2.81 5.57 1.85 1.19 1.18 1.85 5.57 2.81 1.854.72 3.13 3.13

TABLE 19 Paw withdrawal threshold (g) illustrated in FIG. 24. VehicleVehicle Vehicle Vehicle Vehicle Vehicle Vehicle Vehicle Pre-Sx 15 11.6915 15 15 15 15 15 Pre-Drug 7.8 0.65 2.38 1.58 2.81 4.98 4.25 3.67 (D1) 3hrs 5.56 1.19 2.37 0.99 2.2 1.85 2.81 3.13 5 hrs 3.73 1.4 3.33 2.37 1.562.37 8.44 2.81 Pre-Drug 3.67 0.99 2.2 3.73 1.85 3.58 1.85 3.73 (D3) 3hrs 4.25 1.65 7.94 2.38 1.99 1.65 2.37 1.65 5 hrs 6.58 0.44 2.2 3.133.33 1.85 4.25 2.2 compound 2 15 15 15 15 15 15 15 15 6.58 4.4 2.75 1.42.81 5.04 1.18 2.81 4.23 10.04 6.64 3.13 6.64 5.04 1.56 5.04 5.04 7.313.55 1.85 3.72 5.04 3.67 3.13 4.25 8.44 2.81 1.4 4.98 6.4 2 3.33 6.586.58 4.47 3.13 6.58 4.25 3.67 4.4 2.81 4.25 6.66 1.85 6.64 4.25 2.816.64 Gabapentin Gabapentin Gabapentin Gabapentin Gabapentin GabapentinGabapentin Gabapentin 15 15 15 15 15 15 15 15 6.64 1.65 1.33 1.56 2.814.23 5.04 2.81 15 5.18 15 3.67 15 15 6.58 5.56 10.31 5.56 6.58 1.99 6.667.94 6.4 6.58 6.64 1.56 1.4 1.65 5.57 3.55 3.58 2.81 15 15 15 3.33 11.698.61 6.58 9.86 15 4.4 4.72 2.37 6.58 15 7.31 6.66

TABLE 20 Paw threshold (g) illustrated in FIG. 25. Vehicle VehicleVehicle Vehicle Vehicle Vehicle Vehicle Vehicle Pre-Sx 15 15 15 15 15 1515 15 1 wk 6.42 4.25 3.33 4.25 5.57 5.37 9.86 13.96 2 wks 6.66 4.25 0.41.85 2.81 2.2 2.81 6.42 compound 2 15 15 15 15 15 15 15 15 1.85 1.182.81 4.47 6.4 2.2 15 4.25 1.65 2.81 2 2 3.73 0.99 5.56 5.04 GabapentinGabapentin Gabapentin Gabapentin Gabapentin Gabapentin GabapentinGabapentin 15 15 15 15 15 15 15 15 1.56 1.56 6.58 3.73 2.2 6.58 4.25 1.40.99 1.31 3.13 1.4 2.81 2.81 1.4 2.81

TABLE 21 Paw threshold (g) illustrated in FIG. 26. Vehi- Vehi- Vehi-Vehi- cle cle cle Vehicle Vehicle Vehicle Vehicle cle Pre- 15 15 11.6915 15 15 15 15 Sx 1 wk 15 1.85 3.33 5.56 15 15 6.66 4.25 2 wks 7.94 3.336.64 6.66 6.42 15 3.67 2.81 3 wks 15 4.25 2.2 2.2 2.64 6.66 2.81 3.13compound 2 15 15 15 15 15 11.69 15 15 15 11.41 15 3.13 1.18 15 6.66 1513.96 4.25 5.37 11.69 2.8 8.44 7.94 13.96 7.53 6.64 4.98 2.2 4.4 2.3810.31 15

Spinal nerve ligation resulted in the development of tactile allodyniaas indicated by a decreased paw withdrawal threshold to a non-noxioustactile mechanical stimulus, 3 to 4 weeks post-surgery (FIGS. 23-26).Using non-fasted animals, Compound 2 (10 and 50 mg/kg, p.o.) did notproduce a statistically significant difference from vehicle treatedanimals at any time point after any of the three administrations (FIG.23). In separate experiments, gabapentin (100 mg/kg i.p.), the positivecontrol, consistently produced a statistically significant reversal ofallodynia (e.g. FIG. 24)

In a follow up study using animals fasted overnight prior to compoundadministration, Compound 2 (50 mg/kg, p.o.) produced statisticallysignificant dose- and time dependent reversal of allodynia 3 hr postdosing upon first administration. However the magnitude of the effectwas small (FIG. 24), and upon subsequent administrations (48 hrs later)Compound 2 failed to produce a statistically significant effect. Incontrast, gabapentin (100 mg/kg, i.p.), the positive control, produced astatistically significant reversal of hyperalgesia 3 and 5 hours aftereach administration.

When Compound 2 (30 mg/kg, p.o.) was given as a single administrationimmediately following the surgery and behaviorally assessed 1 and 2weeks later a significant difference between treated animals and vehiclecontrols was not noted. Gabapentin when dosed on only the day of surgeryalso failed to alter the course of development of allodynia (FIG. 25).In contrast, when Compound 2 (30 mg/kg, p.o.) was administeredrepeatedly, once a day for 5 days starting on the day of surgery, asignificant reduction in tactile allodynia was noticed 1, 2 and 3 weekspost surgery (FIG. 26).

Overall, Compound 2 (50 mg/kg, p.o.) failed to produce robust andsignificant reversal of tactile allodynia when given as a singleadministration 3 weeks post-surgery. In contrast, when given repeatedlyduring the development of allodynia (QD, days 1-5 post-surgery, 30mg/kg, p.o.) Compound 2 caused a significant reduction in tactileallodynia as compared to vehicle treated controls. These data suggestedthat Compound 2 does not have acute efficacy in the treatment ofestablished neuropathic pain. However, it may affect the time courseand/or magnitude of allodynia during the development phase, i.e.,Compound 2 may delay onset or reduce incidence or severity ofneuropathic pain.

Experimental Example #18 Effect of Compound 2 on Latency to Fall in anAccelerating Rotarod Assay of Ataxia

Compound 2 was evaluated in a rat accelerating rotarod model of ataxia.Ataxia is a common clinical problem with CNS-active compounds and canoften confound interpretation of efficacy in preclinical pain models.The rotarod assay of ataxia has been widely used to assess theside-effect liability of novel pharmacological treatments (Jones &Roberts, 1968, J. Pharm. Pharmacol. 20:302-04). The effect of Compound 2on latency to fall in an accelerating rotarod assay of ataxia in ratswas evaluated. In one aspect, the data could be used to establish atherapeutic index for Compound 2 vs. ataxia/sedation.

The positive control was haloperidol (Sigma, St. Louis), and thenegative control was vehicle (which was HPMethylCellulose for Compound2; and 15% DMA, 65% PEG300, and 20% D5W for haloperidol). MaleSprague-Dawley Rats (Harlan, Ill) were 250-300 g at time of dosing.Rotarod was obtained from IITC, CA.

To examine the potential effects of Compound 2 on motor performance,rats were tested on an accelerating rotarod (IITC, Ca). In this assay,rats were placed on the rotarod with the speed set to accelerate from 4to 40 rpm over 300 seconds. The maximum time spent on the rotarod wasset at 300 seconds. Rats received two timed training trials (averaged togive the reported baseline) on the first day, then 24 hr later rats wereadministered Compound 2 (30, 100 and 300 mg/kg, p.o.), haloperidol (3mg/kg, p.o.), the positive control, or vehicle. Latency to fall wasassessed 1 hr post-drug administration corresponding to the T_(max) forCompound 2 in rats when given orally.

Statistical significance was determined on untransformed data using atwo-way analysis of variance (Graphpad Prism, NC). Significant maineffects were analyzed further by subsequent Bonferronni post-hoc test.The level of significance was set at p<0.05. Data are shown asmean±S.E.M. (with an asterisk to denote significance as compared tovehicle treated controls).

Table 22 summarizes the in vivo pharmacology data. Table 23 summarizesthe latency to fall data illustrated in FIG. 27.

TABLE 22 In vivo pharmacology data. Method and Gender and Species/ Timeof Doses No. per Noteworthy Test System Strain Administration (units)Group Findings Rotarod Rat/SD p.o. 30, 100, M, n = 11- Compound 2 didnot 300 mg/kg 12 significantly reduce latency to fall

TABLE 23 Latency to fall (sec) illustrated in FIG. 27. Vehicle VehicleVehicle Vehicle Vehicle Vehicle Vehicle Vehicle Baseline 44.5 51.5 6964.5 50.5 62 72 78.5 Post- 37 127 118 89 87 91 179 140 Drug compound 2(300 mg/kg) Baseline 84 76.5 57 86.5 81.5 79 71.5 86.5 Post- 184 55 11255 105 184 124 175 Drug compound 2 Vehicle Vehicle Vehicle (30 mg/kg)Baseline 105.5 46 64 70 108.5 72 68 Post- 118 105 80 39 99 72 94 Drugcompound 2 (300 mg/kg) Haloperidol Haloperidol Haloperidol HaloperidolBaseline 61 97.5 89 60 38 42.5 59 Post- 43 131 78 9 10 7 2 Drug compound2 (30 mg/kg) Baseline 52 49 60.5 48.5 47.5 68 47.5 97 Post- 66 81 133120 56 115 50 70 Drug Haloperidol Haloperidol Haloperidol HaloperidolHaloperidol Haloperidol Haloperidol 3 mg/kg Baseline 68 48 86 62 71 8057 Post- 5 5 6 2 30 10 15 Drug compound 2 (100 mg/kg) Baseline 43.5 75.555 63 60.5 73.5 Post- 86 64 45 111 91 221 Drug Baseline Post- Drugcompound 2 (100 mg/kg) Baseline 73 72 54.5 54 46.5 53 Post- 72 45 66 2072 199 Drug Baseline Post- Drug

Latency to fall increased in all groups between baseline and post-drugeffects; performance improves with repeated runs on the rotarod.Compound 2 (30, 100 and 300 mg/kg, p.o.) did not significantly decreaselatency to fall in the rotarod assay at 1 hr post-dosing as compared tovehicle treated animals. In contrast, haloperidol (3 mg/kg, p.o.), thepositive control, resulted in significant motor deficits 1 hpost-administration (FIG. 27).

The present study evaluated Compound 2 in an rat accelerating rotarodassay, a model of ataxia and sedation. Compound 2 (30-300 mg/kg, p.o.)did not significantly reduce latency to fall 1 hr post-dosing. Incontrast, haloperidol (3 mg/kg, p.o.) produced a substantial reductionin latency. These data suggested that high doses of Compound 2 did notcause ataxia in rats. This in turn suggested that efficacy signals seenacross multiple pain models were not due to confounding ataxia andsedation. The data also suggested that Compound 2 did not cause ataxiaand sedation clinically. This is consistent with Compound 2 havinglimited CNS penetration, and suggested that the primary site of actionfor the analgesic effects of GAL-044 may be in the periphery.

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety.

While this invention has been disclosed with reference to specificembodiments, it is apparent that other embodiments and variations ofthis invention may be devised by others skilled in the art withoutdeparting from the true spirit and scope of the invention. The appendedclaims are intended to be construed to include all such embodiments andequivalent variations.

1. A compound according to Formula I or a salt thereof:

wherein: R¹ and R² are independently hydrogen or alkyl, or R¹ and R²form together a radical according to the formula (CH₂)_(n), wherein2≦n≦6; R³ is, independently at each occurrence, hydrogen, alkyl,substituted alkyl, alkenyl, alkynyl, halogen, phenyl, substitutedphenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl,hydroxyl, alkoxy, cyano, nitro, acyl, carboxy, carboxyalkyl, or amido;R⁴ is hydrogen, alkyl, substituted alkyl, or acyl; and W is hydrogen,alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl,carboxy, or carboxyalkyl.
 2. The compound of claim 1, wherein R¹ and R²are CH₃.
 3. The compound of claim 1, wherein the compound according toFormula I is selected from the group consisting of(S)-2-(2-hydroxyphenylamino)-thiazoline-5,5-dimethyl-4-carboxylic acid,(R)-2-(2-hydroxyphenylamino)-thiazoline-5,5-dimethyl-4-carboxylic acid,(R)-2-(2-Hydroxy-4-methoxyphenylamino)-thiazoline-4-carboxylic acid,(R)-2-(4-Fluoro-2-hydroxyphenylamino)-thiazoline-4-carboxylic acid,(R)-2-(3,5-Dichloro-2-hydroxy-4-methylphenylamino)-thiazoline-4-carboxylicacid, (R)-2-(5-tert-Butyl-2-hydroxyphenylamino)-thiazoline-4-carboxylicacid,(R)-2-(2-Hydroxy-4-methoxycarbonylphenylamino)-thiazoline-4-carboxylicacid,(R)-2-(5-Ethanesulfonyl-2-hydroxy-phenylamino)-thiazoline-4-carboxylicacid, (R)-2-(4-Chloro-2-hydroxyphenylamino)-thiazoline-4-carboxylicacid, (R)-2-(2-Hydroxy-5-methoxyphenylamino)-thiazoline-4-carboxylicacid,(S)-2-(2-Hydroxy-5-chlorophenylamino)-thiazoline-5,5-dimethyl-4-carboxylicacid methyl ester,(S)-2-(2-Hydroxy-5-chlorophenylamino)-thiazoline-5,5-dimethyl-4-carboxylicacid,(S)-2-(2-Hydroxy-5-methylphenylamino)-thiazoline-5,5-dimethyl-4-carboxylicacid methyl ester,(S)-2-(2-Hydroxy-5-methylphenylamino)-thiazoline-5,5-dimethyl-4-carboxylicacid,(S)-2-(2-Hydroxy-4-methoxyphenylamino)-thiazoline-5,5-dimethyl-4-carboxylicacid,(S)-2-(2-Hydroxy-4-methoxyphenylamino)-thiazoline-5,5-dimethyl-4-carboxylicacid methyl ester,(S)-2-(4-Chloro-2-hydroxyphenylamino)-thiazoline-5,5-dimethyl-4-carboxylicacid,(S)-2-(2-Hydroxy-5-nitrophenylamino)-thiazoline-5,5-dimethyl-4-carboxylicacid methyl ester,(S)-2-(2-Hydroxy-5-nitrophenylamino)-thiazoline-5,5-dimethyl-4-carboxylicacid,(S)-2-(5-Ethanesulfonyl-2-hydroxyphenylamino)-thiazoline-5,5-dimethyl-4-carboxylicacid methyl ester,(S)-2-(5-Ethanesulfonyl-2-hydroxyphenylamino)-thiazoline-5,5-dimethyl-4-carboxylicacid, (R)-2-(2-hydroxyphenylamino)-thiazoline-4-carboxylic acid methylester, (S)-2-(2-hydroxyphenylamino)-thiazoline-5,5-dimethyl-4-carboxylicacid methyl ester,(R)-2-(4-chloro-2-hydroxyphenylamino)-thiazoline-4-carboxylic acidmethyl ester,(S)-2-(4-chloro-2-hydroxyphenylamino)-thiazoline-5,5-dimethyl-4-carboxylicacid methyl ester,(R)-2-(4-chloro-2-hydroxyphenylamino)-thiazoline-4-carboxylic acid, and(R)-2-(2-hydroxyphenylamino)-thiazoline-5,5-dimethyl-4-carboxylic acidmethyl ester, mixtures thereof and salts thereof.
 4. A method of makinga compound according to Formula I or a salt thereof, said methodcomprising: a) reacting a compound according to formula A:

with a compound according to formula C:

to give a compound according to formula D:

 and b) deprotecting the compound according to formula D to give thecompound according to Formula I or a salt thereof; wherein R¹ and R²are, independently, hydrogen or alkyl, or R¹ and R² form together aradical according to the formula (CH₂)_(n), wherein 2≦n≦6; R³ is,independently at each occurrence, hydrogen, alkyl, substituted alkyl,alkenyl, alkynyl, halogen, phenyl, substituted phenyl, aryl, substitutedaryl, heteroaryl, substituted heteroaryl, hydroxyl, alkoxy, cyano,nitro, acyl, carboxy, carboxyalkyl, or amido; R⁴ is hydrogen, alkyl,substituted alkyl, or acyl; W is hydrogen, alkyl, aryl, substitutedaryl, heteroaryl, substituted heteroaryl, carboxy, or carboxyalkyl; Z isCl, Br, or I; and P is a thiol protecting group.
 5. A method of making acompound according to Formula I or a salt thereof, said methodcomprising: reacting a compound according to formula A:

with a compound according to formula B:

to give the compound according to Formula I or a salt thereof; whereinR¹ and R² are independently hydrogen or alkyl, or R¹ and R² formtogether a radical according to the formula (CH₂)_(n), wherein 2≦n≦6; R³is, independently at each occurrence, hydrogen, alkyl, substitutedalkyl, alkenyl, alkynyl, halogen, phenyl, substituted phenyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, hydroxyl, alkoxy,cyano, nitro, acyl, carboxy, carboxyalkyl, or amido; R⁴ is hydrogen,alkyl, substituted alkyl, or acyl; W is hydrogen, alkyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, carboxy, orcarboxyalkyl; and Z is Cl, Br, or I.
 6. A method of diminishing thedosage of a narcotic analgesic required to achieve pain relief in asubject in need thereof, the method comprising the step of administeringto the subject a therapeutically effective amount of at least onecompound according to Formula I or a salt thereof,

wherein: R¹ and R² are independently hydrogen or alkyl, or R¹ and R²form together a radical according to the formula (CH₂)_(n), wherein2≦n≦6; R³ is, independently at each occurrence, hydrogen, alkyl,substituted alkyl, alkenyl, alkynyl, halogen, phenyl, substitutedphenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl,hydroxyl, alkoxy, cyano, nitro, acyl, carboxy, carboxyalkyl, or amido;R⁴ is hydrogen, alkyl, substituted alkyl, or acyl; and W is hydrogen,alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl,carboxy, or carboxyalky, whereby the dosage of the narcotic analgesicrequired to achieve pain relief in the subject is diminished.
 7. Themethod of claim 6, wherein the compound is administered in a therapeuticdosing regimen or a preconditioning dosing regimen.
 8. The method ofclaim 6, wherein the subject is a human.
 9. A method of extending theduration of action or analgesic efficacy of a narcotic analgesic in asubject in need thereof, the method comprising the step of administeringto the subject a therapeutically effective amount of at least onecompound according to Formula I or a salt thereof,

wherein: R¹ and R² are independently hydrogen or alkyl, or R¹ and R²form together a radical according to the formula (CH₂)_(n), wherein2≦n≦6; R³ is, independently at each occurrence, hydrogen, alkyl,substituted alkyl, alkenyl, alkynyl, halogen, phenyl, substitutedphenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl,hydroxyl, alkoxy, cyano, nitro, acyl, carboxy, carboxyalkyl, or amido;R⁴ is hydrogen, alkyl, substituted alkyl, or acyl; and W is hydrogen,alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl,carboxy, or carboxyalky, whereby the duration of action or analgesicefficacy of the narcotic analgesic in the subject is extended.
 10. Themethod of claim 9, wherein the compound is administered in a therapeuticdosing regimen or a preconditioning dosing regimen.
 11. The method ofclaim 9, wherein the subject is a human.
 12. A method of preventingtachyphylaxis in a subject in need thereof, the method comprising thestep of administering to the subject a therapeutically effective amountof at least one compound according to Formula I or a salt thereof,

wherein: R¹ and R² are independently hydrogen or alkyl, or R¹ and R²form together a radical according to the formula (CH₂)_(n), wherein2≦n≦6; R³ is, independently at each occurrence, hydrogen, alkyl,substituted alkyl, alkenyl, alkynyl, halogen, phenyl, substitutedphenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl,hydroxyl, alkoxy, cyano, nitro, acyl, carboxy, carboxyalkyl, or amido;R⁴ is hydrogen, alkyl, substituted alkyl, or acyl; W is hydrogen, alkyl,aryl, substituted aryl, heteroaryl, substituted heteroaryl, carboxy, orcarboxyalkyl, whereby tachyphylaxis in the subject is prevented.
 13. Themethod of claim 12, wherein the compound is administered in atherapeutic dosing regimen or a preconditioning dosing regimen.
 14. Themethod of claim 12, wherein the subject is a human.
 15. A method ofreducing the level of activity of reactive oxygen species and reactivenitrogen species in a subject in need thereof, the method comprising thestep of administering to the subject a therapeutically effective amountof at least one compound according to Formula I or a salt thereof,

wherein: R¹ and R² are independently hydrogen or alkyl, or R¹ and R²form together a radical according to the formula (CH₂)_(n), wherein2≦n≦6; R³ is, independently at each occurrence, hydrogen, alkyl,substituted alkyl, alkenyl, alkynyl, halogen, phenyl, substitutedphenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl,hydroxyl, alkoxy, cyano, nitro, acyl, carboxy, carboxyalkyl, or amido;R⁴ is hydrogen, alkyl, substituted alkyl, or acyl; and W is hydrogen,alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl,carboxy, or carboxyalkyl, whereby the level of activity of reactiveoxygen species and reactive nitrogen species in the subject is reduced.16. The method of claim 15, wherein the reactive oxygen species isperoxynitrite.
 17. The method of claim 15, wherein the subject exhibitssymptoms of at least one disorder from the group consisting of: stroke,myocardial infarction, chronic heart failure, circulatory shock, chronicinflammatory diseases, cancer, neurogenerative disorders, sleep apnea,diabetes, narcotic analgesic tachyphylaxis, pancreatitis pain, addictivebehavior, and hypertension.
 18. The method of claim 15, wherein thecompound is administered in a therapeutic dosing regimen or apreconditioning dosing regimen.
 19. The method of claim 15, wherein thelevel of activity of reactive oxygen species is reduced by diminishingreactive oxygen species production.
 20. The method of claim 15, whereinthe subject is a human.